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Francis D, Bhairaddy A, Joy A, Hari GV, Francis A. Secretory proteins in the orchestration of microbial virulence: The curious case of Staphylococcus aureus. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2023; 133:271-350. [PMID: 36707204 DOI: 10.1016/bs.apcsb.2022.10.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Microbial virulence showcases an excellent model for adaptive changes that enable an organism to survive and proliferate in a hostile environment and exploit host resources to its own benefit. In Staphylococcus aureus, an opportunistic pathogen of the human host, known for the diversity of the disease conditions it inflicts and the rapid evolution of antibiotic resistance, virulence is a consequence of having a highly plastic genome that is amenable to quick reprogramming and the ability to express a diverse arsenal of virulence factors. Virulence factors that are secreted to the host milieu effectively manipulate the host conditions to favor bacterial survival and growth. They assist in colonization, nutrient acquisition, immune evasion, and systemic spread. The structural and functional characteristics of the secreted virulence proteins have been shaped to assist S. aureus in thriving and disseminating effectively within the host environment and exploiting the host resources to its best benefit. With the aim of highlighting the importance of secreted virulence proteins in bacterial virulence, the present chapter provides a comprehensive account of the role of the major secreted proteins of S. aureus in orchestrating its virulence in the human host.
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Affiliation(s)
- Dileep Francis
- Department of Life Sciences, Kristu Jayanti College, Autonomous, Bengaluru, Karnataka, India.
| | - Anusha Bhairaddy
- Department of Life Sciences, Kristu Jayanti College, Autonomous, Bengaluru, Karnataka, India
| | - Atheene Joy
- Department of Life Sciences, Kristu Jayanti College, Autonomous, Bengaluru, Karnataka, India
| | | | - Ashik Francis
- Rajiv Gandhi Centre for Biotechnology, Trivandrum, Kerala, India
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2
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Claussin C, Vazquez J, Whitehouse I. Single-molecule mapping of replisome progression. Mol Cell 2022; 82:1372-1382.e4. [PMID: 35240057 PMCID: PMC8995386 DOI: 10.1016/j.molcel.2022.02.010] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2021] [Revised: 12/22/2021] [Accepted: 02/02/2022] [Indexed: 10/19/2022]
Abstract
Fundamental aspects of DNA replication, such as the anatomy of replication stall sites, how replisomes are influenced by gene transcription, and whether the progression of sister replisomes is coordinated, are poorly understood. Available techniques do not allow the precise mapping of the positions of individual replisomes on chromatin. We have developed a method called Replicon-seq that entails the excision of full-length replicons by controlled nuclease cleavage at replication forks. Replicons are sequenced using Nanopore, which provides a single-molecule readout of long DNA. Using Replicon-seq, we found that sister replisomes function autonomously and yet progress through chromatin with remarkable consistency. Replication forks that encounter obstacles pause for a short duration but rapidly resume synthesis. The helicase Rrm3 plays a critical role both in mitigating the effect of protein barriers and with facilitating efficient termination. Replicon-seq provides a high-resolution means of defining how individual replisomes move across the genome.
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Affiliation(s)
- Clémence Claussin
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
| | - Jacob Vazquez
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
| | - Iestyn Whitehouse
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
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3
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Wiemels RE, Keogh RA, Carroll RK. Detection and Quantification of Secreted Nuclease Activity in Staphylococcus aureus Culture Supernatants. Methods Mol Biol 2021; 2341:17-24. [PMID: 34264456 PMCID: PMC10152981 DOI: 10.1007/978-1-0716-1550-8_3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
Staphylococcal secreted nuclease contributes to S. aureus virulence by degrading neutrophil extracellular traps (NETs), which allows the bacterium to evade the host immune system and has also been shown to promote biofilm dispersal. In this chapter, two methods for detecting nuclease activity are described, both of which have increased sensitivity compared to the traditional nuclease agar method.
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Affiliation(s)
| | - Rebecca A Keogh
- Department of Biological Sciences, Ohio University, Athens, OH, USA
| | - Ronan K Carroll
- Department of Biological Sciences, Ohio University, Athens, OH, USA.
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Soh KY, Loh JMS, Hall C, Proft T. Functional Analysis of Two Novel Streptococcus iniae Virulence Factors Using a Zebrafish Infection Model. Microorganisms 2020; 8:E1361. [PMID: 32899555 PMCID: PMC7564053 DOI: 10.3390/microorganisms8091361] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2020] [Revised: 08/31/2020] [Accepted: 09/03/2020] [Indexed: 12/30/2022] Open
Abstract
Streptococcus iniae is a major fish pathogen that contributes to large annual losses in the aquaculture industry, exceeding US$100 million. It is also reported to cause opportunistic infections in humans. We have recently identified two novel S. iniae virulence factors, an extracellular nuclease (SpnAi) and a secreted nucleotidase (S5nAi), and verified their predicted enzymatic activities using recombinant proteins. Here, we report the generation of green fluorescent S. iniae spnAi and s5nAi deletion mutants and their evaluation in a transgenic zebrafish infection model. Our results show nuclease and nucleotidase activities in S. iniae could be attributed to SpnAi and S5nAi, respectively. Consistent with this, larvae infected with the deletion mutants demonstrated enhanced survival and bacterial clearance, compared to those infected with wild-type (WT) S. iniae. Deletion of spnAi and s5nAi resulted in sustained recruitment of neutrophils and macrophages, respectively, to the site of infection. We also show that recombinant SpnAi is able to degrade neutrophil extracellular traps (NETs) isolated from zebrafish kidney tissue. Our results suggest that both enzymes play an important role in S. iniae immune evasion and might present potential targets for the development of therapeutic agents or vaccines.
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Affiliation(s)
- Kar Yan Soh
- Department of Molecular Medicine & Pathology, School of Medical Sciences, The University of Auckland, Auckland 1142, New Zealand; (K.Y.S.); (J.M.S.L.)
- Maurice Wilkins Centre for Biomolecular Discoveries, The University of Auckland, Auckland 1142, New Zealand
| | - Jacelyn Mei San Loh
- Department of Molecular Medicine & Pathology, School of Medical Sciences, The University of Auckland, Auckland 1142, New Zealand; (K.Y.S.); (J.M.S.L.)
- Maurice Wilkins Centre for Biomolecular Discoveries, The University of Auckland, Auckland 1142, New Zealand
| | - Christopher Hall
- Department of Molecular Medicine & Pathology, School of Medical Sciences, The University of Auckland, Auckland 1142, New Zealand; (K.Y.S.); (J.M.S.L.)
| | - Thomas Proft
- Department of Molecular Medicine & Pathology, School of Medical Sciences, The University of Auckland, Auckland 1142, New Zealand; (K.Y.S.); (J.M.S.L.)
- Maurice Wilkins Centre for Biomolecular Discoveries, The University of Auckland, Auckland 1142, New Zealand
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Bai M, Liang M, Huai B, Gao H, Tong P, Shen R, He H, Wu H. Ca2+-dependent nuclease is involved in DNA degradation during the formation of the secretory cavity by programmed cell death in fruit of Citrus grandis 'Tomentosa'. JOURNAL OF EXPERIMENTAL BOTANY 2020; 71:4812-4827. [PMID: 32324220 PMCID: PMC7410178 DOI: 10.1093/jxb/eraa199] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Accepted: 04/21/2020] [Indexed: 05/09/2023]
Abstract
The secretory cavity is a typical structure in Citrus fruit and is formed by schizolysigeny. Previous reports have indicated that programmed cell death (PCD) is involved in the degradation of secretory cavity cells in the fruit, and that the spatio-temporal location of calcium is closely related to nuclear DNA degradation in this process; however, the molecular mechanisms underlying this Ca2+ regulation remain largely unknown. Here, we identified CgCaN that encodes a Ca2+-dependent DNase in the fruit of Citrus grandis 'Tomentosa', the function of which was studied using calcium ion localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The results suggested that the full-length cDNA of CgCaN contains an ORF of 1011 bp that encodes a protein 336 amino acids in length with a SNase-like functional domain. CgCaN digests dsDNA at neutral pH in a Ca2+-dependent manner. In situ hybridization signals of CgCaN were particularly distributed in the secretory cavity cells. Ca2+ and Ca2+-dependent DNases were mainly observed in the condensed chromatin and in the nucleolus. In addition, spatio-temporal expression patterns of CgCaN and its protein coincided with the time-points that corresponded to chromatin degradation and nuclear rupture during the PCD in the development of the fruit secretory cavity. Taken together, our results suggest that Ca2+-dependent DNases play direct roles in nuclear DNA degradation during the PCD of secretory cavity cells during Citrus fruit development. Given the consistency of the expression patterns of genes regulated by calmodulin (CaM) and calcium-dependent protein kinases (CDPK) and the dynamics of calcium accumulation, we speculate that CaM and CDPK proteins might be involved in Ca2+ transport from the extracellular walls through the cytoplasm and into the nucleus to activate CgCaN for DNA degradation.
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Affiliation(s)
- Mei Bai
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China
| | - Minjian Liang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China
| | - Bin Huai
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China
| | - Han Gao
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China
| | - Panpan Tong
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China
| | - Rongxin Shen
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China
| | - Hanjun He
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China
| | - Hong Wu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China
- Correspondence:
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Trusch F, Loebach L, Wawra S, Durward E, Wuensch A, Iberahim NA, de Bruijn I, MacKenzie K, Willems A, Toloczko A, Diéguez-Uribeondo J, Rasmussen T, Schrader T, Bayer P, Secombes CJ, van West P. Cell entry of a host-targeting protein of oomycetes requires gp96. Nat Commun 2018; 9:2347. [PMID: 29904064 PMCID: PMC6002402 DOI: 10.1038/s41467-018-04796-3] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2017] [Accepted: 05/11/2018] [Indexed: 12/02/2022] Open
Abstract
The animal-pathogenic oomycete Saprolegnia parasitica causes serious losses in aquaculture by infecting and killing freshwater fish. Like plant-pathogenic oomycetes, S. parasitica employs similar infection structures and secretes effector proteins that translocate into host cells to manipulate the host. Here, we show that the host-targeting protein SpHtp3 enters fish cells in a pathogen-independent manner. This uptake process is guided by a gp96-like receptor and can be inhibited by supramolecular tweezers. The C-terminus of SpHtp3 (containing the amino acid sequence YKARK), and not the N-terminal RxLR motif, is responsible for the uptake into host cells. Following translocation, SpHtp3 is released from vesicles into the cytoplasm by another host-targeting protein where it degrades nucleic acids. The effector translocation mechanism described here, is potentially also relevant for other pathogen-host interactions as gp96 is found in both animals and plants.
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Grants
- BB/E007120/1 Biotechnology and Biological Sciences Research Council
- BB/G012075/1 Biotechnology and Biological Sciences Research Council
- Biotechnology and Biological Sciences Research Council (BBSRC)
- Deutsche Forschungsgemeinschaft (German Research Foundation)
- Our work is supported by the [European Community's] Seventh Framework Programme [FP7/2007-2013] under grant agreement no [238550] (LL, JDU, CJS, PvW); BBSRC [BBE007120/1, BB/J018333/1 and BB/G012075/1] (FT, IdB, CJS, SW, PvW); Newton Global partnership Award [BB/N005058/1] (FT, PvW), the University of Aberdeen (ADT, TR, CJS, PvW) and Deutsche Forschungsgemeinschaft [CRC1093] (PB, TS). We would like to acknowledge the Ministry of Higher Education Malaysia for funding INA. We would like to thank Brian Haas for his bioinformatics support. We would like to acknowledge Neil Gow and Johannes van den Boom for critical reading of the manuscript. We would like to acknowledge Svetlana Rezinciuc for technical help with pH-studies.
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Affiliation(s)
- Franziska Trusch
- Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
- International Centre for Aquaculture Research and Development (ICARD), University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
| | - Lars Loebach
- Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
| | - Stephan Wawra
- Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
- Botanical Institute, Genetical Institute, University of Cologne, Cologne, 50674, Germany
| | - Elaine Durward
- Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
- International Centre for Aquaculture Research and Development (ICARD), University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
| | - Andreas Wuensch
- Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
- International Centre for Aquaculture Research and Development (ICARD), University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
| | - Nurul Aqilah Iberahim
- Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
- International Centre for Aquaculture Research and Development (ICARD), University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
- School of Fisheries and Aquaculture Sciences, Universiti Malaysia Terengganu, 21030, Kuala Terengganu, Terengganu, Malaysia
| | - Irene de Bruijn
- Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
- Netherlands Institute for Ecology (NIOO), Wageningen, 6708 PB, Netherlands
| | - Kevin MacKenzie
- Microscopy and Histology Facility, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
| | - Ariane Willems
- Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
| | - Aleksandra Toloczko
- Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
| | | | - Tim Rasmussen
- Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
| | - Thomas Schrader
- Organic Chemistry, University of Duisburg-Essen, Essen, 45117, Germany
| | - Peter Bayer
- Structural and Medicinal Biochemistry, Centre for Medical Biotechnology (ZMB), University of Duisburg-Essen, Essen, 45117, Germany
| | - Chris J Secombes
- International Centre for Aquaculture Research and Development (ICARD), University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK
- Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, Scotland, UK
| | - Pieter van West
- Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK.
- International Centre for Aquaculture Research and Development (ICARD), University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK.
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Effect of high-pressure treatment on taste and metabolite profiles of ducks with two different vinasse-curing processes. Food Res Int 2017; 105:703-712. [PMID: 29433265 DOI: 10.1016/j.foodres.2017.11.084] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2017] [Revised: 11/23/2017] [Accepted: 11/30/2017] [Indexed: 01/23/2023]
Abstract
The effect of high-pressure (HP) (0.1, 150 and 300MPa, 15min) on taste profiles of vinasse-cured ducks was investigated; the metabolite profiles were determined using 1H NMR. HP at 150MPa increased the taste intensity of products compared with the controls, while HP at 300MPa did not further improve their taste compared with 150MPa treated samples. The metabonome of vinasse-cured ducks was dominated by 27 metabolites. HP increased amino acids, glucose, alkaloids and organic acids, but decreased inosine monophosphate and its derivatives, compared with the controls. The increments of metabolites in vinasse-dry-cured duck were higher than those in vinasse-wet-cured duck. The change of metabolites could be related to the enzyme activity, the degradations of proteins, sugars and nucleotides, and the permeation from vinasse-curing agents to duck meat. These findings suggest that 150MPa treatment was effective to improve the taste of vinasse-cured duck.
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8
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An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease. J Bacteriol 2016; 199:JB.00453-16. [PMID: 27795319 DOI: 10.1128/jb.00453-16] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2016] [Accepted: 10/04/2016] [Indexed: 12/14/2022] Open
Abstract
Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.
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Kiedrowski MR, Crosby HA, Hernandez FJ, Malone CL, McNamara JO, Horswill AR. Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease. PLoS One 2014; 9:e95574. [PMID: 24752186 PMCID: PMC3994088 DOI: 10.1371/journal.pone.0095574] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2013] [Accepted: 03/28/2014] [Indexed: 12/30/2022] Open
Abstract
Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.
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Affiliation(s)
- Megan R. Kiedrowski
- Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America
| | - Heidi A. Crosby
- Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America
| | - Frank J. Hernandez
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America
| | - Cheryl L. Malone
- Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America
| | - James O. McNamara
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America
| | - Alexander R. Horswill
- Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America
- * E-mail:
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10
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Xie T, Feng Y, Shan L, Wang J. Modeling of the [E43S]SNase-ssDNA–Cd2+ complex: Structural insight into the action of nuclease on ssDNA. Arch Biochem Biophys 2013; 532:103-13. [DOI: 10.1016/j.abb.2013.02.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2012] [Revised: 02/04/2013] [Accepted: 02/06/2013] [Indexed: 11/30/2022]
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11
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Hu Y, Meng J, Shi C, Hervin K, Fratamico PM, Shi X. Characterization and comparative analysis of a second thermonuclease from Staphylococcus aureus. Microbiol Res 2013; 168:174-82. [DOI: 10.1016/j.micres.2012.09.003] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2012] [Revised: 09/12/2012] [Accepted: 09/14/2012] [Indexed: 12/12/2022]
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12
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Chen HM, Pang Y, Zeng J, Ding Q, Yin SY, Liu C, Lu MZ, Cui KM, He XQ. The Ca2+ -dependent DNases are involved in secondary xylem development in Eucommia ulmoides. JOURNAL OF INTEGRATIVE PLANT BIOLOGY 2012; 54:456-70. [PMID: 22694768 DOI: 10.1111/j.1744-7909.2012.01134.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/14/2023]
Abstract
Secondary xylem development has long been recognized as a typical case of programmed cell death (PCD) in plants. During PCD, the degradation of genomic DNA is catalyzed by endonucleases. However, to date, no endonuclease has been shown to participate in secondary xylem development. Two novel Ca(2+) -dependent DNase genes, EuCaN1 and EuCaN2, were identified from the differentiating secondary xylem of the tree Eucommia ulmoides Oliv., their functions were studied by DNase activity assay, in situ hybridization, protein immunolocalization and virus-induced gene silencing experiments. Full-length cDNAs of EuCaN1 and EuCaN2 contained an open reading frame of 987 bp, encoding two proteins of 328 amino acids with SNase-like functional domains. The genomic DNA sequence for EuCaN1 had no introns, while EuCaN2 had 8 introns. EuCaN1 and EuCaN2 digested ssDNA and dsDNA with Ca(2+) -dependence at neutral pH. Their expression was confined to differentiating secondary xylem cells and the proteins were localized in the nucleus. Their activity dynamics was closely correlated with secondary xylem development. Secondary xylem cell differentiation is influenced by RNAi of endonuclease genes. The results provide evidence that the Ca(2+) -dependent DNases are involved in secondary xylem development.
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Affiliation(s)
- Hui-Min Chen
- State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China
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Abstract
Streptomyces nucleases are widely distributed and multifunctional enzymes acting on both DNA and RNA. They occur extra as well as intracellularly and can be classified under sugar specific and sugar non-specific nucleases. Nucleases play different roles like analytical, biological, and nutritional. They are also used in programmed cell death. Although more than 20 nucleases are reported to date, very little information is available regarding their structure-function relationship, active site based sequence homology, and the probable mechanism of action. This review describes the history, occurrence, localization, production, purification, properties, and applications of Streptomyces nucleases.
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Affiliation(s)
- Amruta Pramod Joshi
- National Chemical Laboratory, Division of Biochemical Sciences, Dr. Homi Bhabha Road, Pashan, Pune, India
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14
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Wang CC, Tsong TY, Hsu YH, Marszalek PE. Inhibitor binding increases the mechanical stability of staphylococcal nuclease. Biophys J 2011; 100:1094-9. [PMID: 21320455 DOI: 10.1016/j.bpj.2011.01.011] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2010] [Revised: 12/20/2010] [Accepted: 01/03/2011] [Indexed: 12/19/2022] Open
Abstract
Staphylococcal nuclease (SNase) catalyzes the hydrolysis of DNA and RNA in a calcium-dependent fashion. We used AFM-based single-molecule force spectroscopy to investigate the mechanical stability of SNase alone and in its complex with an SNase inhibitor, deoxythymidine 3',5'-bisphosphate. We found that the enzyme unfolds in an all-or-none fashion at ∼26 pN. Upon binding to the inhibitor, the mechanical unfolding forces of the enzyme-inhibitor complex increase to ∼50 pN. This inhibitor-induced increase in the mechanical stability of the enzyme is consistent with the increased thermodynamical stability of the complex over that of SNase. Because of its strong mechanical response to inhibitor binding, SNase, a model protein folding system, offers a unique opportunity for studying the relationship between enzyme mechanics and catalysis.
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Affiliation(s)
- Chien-Chung Wang
- Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan, Republic of China. [corrected]
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15
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Simultaneous detection of Staphylococcus aureus enterotoxin C-producing strains from clinical and environmental samples by multiplex PCR assay. ANN MICROBIOL 2010. [DOI: 10.1007/s13213-010-0176-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
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16
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Abstract
Nucleases cleave the phosphodiester bonds of nucleic acids and may be endo or exo, DNase or RNase, topoisomerases, recombinases, ribozymes, or RNA splicing enzymes. In this review, I survey nuclease activities with known structures and catalytic machinery and classify them by reaction mechanism and metal-ion dependence and by their biological function ranging from DNA replication, recombination, repair, RNA maturation, processing, interference, to defense, nutrient regeneration or cell death. Several general principles emerge from this analysis. There is little correlation between catalytic mechanism and biological function. A single catalytic mechanism can be adapted in a variety of reactions and biological pathways. Conversely, a single biological process can often be accomplished by multiple tertiary and quaternary folds and by more than one catalytic mechanism. Two-metal-ion-dependent nucleases comprise the largest number of different tertiary folds and mediate the most diverse set of biological functions. Metal-ion-dependent cleavage is exclusively associated with exonucleases producing mononucleotides and endonucleases that cleave double- or single-stranded substrates in helical and base-stacked conformations. All metal-ion-independent RNases generate 2',3'-cyclic phosphate products, and all metal-ion-independent DNases form phospho-protein intermediates. I also find several previously unnoted relationships between different nucleases and shared catalytic configurations.
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17
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Casero MS, Sanchez-Montero JM, Sinisterra JV. DNase Activity in Immobilized Derivatives of Micrococcal Endonuclease in Merrifield Resin. ACTA ACUST UNITED AC 2009. [DOI: 10.3109/10242428909003653] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Affiliation(s)
- M. Soledad Casero
- Organic Chemistry Department., Faculty of Sciences, 14004, Córdoba, Spain
| | | | - Jose V. Sinisterra
- Organic and Pharmaceutical Chemistry Department., Faculty of Pharmacy, Universidad Complutense de Madrid, 28040, Madrid, Spain
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18
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Watson E, Matousek WM, Irimies EL, Alexandrescu AT. Partially folded states of staphylococcal nuclease highlight the conserved structural hierarchy of OB-fold proteins. Biochemistry 2007; 46:9484-94. [PMID: 17661445 PMCID: PMC2128864 DOI: 10.1021/bi700532j] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
We have been interested in whether three proteins that share a five-stranded beta-barrel "OB-fold" structural motif but no detectable sequence homology fold by similar mechanisms. Here we describe native-state hydrogen exchange experiments as a function of urea for SN (staphylococcal nuclease), a protein with an OB-fold motif and additional nonconserved elements of structure. The regions of structure with the largest stability and unfolding cooperativity are contained within the conserved OB-fold portion of SN, consistent with previous results for CspA (cold shock protein A) and LysN (anticodon binding domain of lysyl tRNA synthetase). The OB-fold also has the subset of residues with the slowest unfolding rates in the three proteins, as determined by hydrogen exchange experiments in the EX1 limit. Although the protein folding hierarchy is maintained at the level of supersecondary structure, it is not evident for individual residues as might be expected if folding depended on obligatory nucleation sites. Rather, the site-specific stability profiles appear to be linked to sequence hydrophobicity and to the density of long-range contacts at each site in the three-dimensional structures of the proteins. We discuss the implications of the correlation between stability to unfolding and conservation of structure for mechanisms of protein structure evolution.
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Affiliation(s)
| | | | | | - Andrei T. Alexandrescu
- * To whom correspondence should be addressed: Department of Molecular and Cell Biology, University of Connecticut, 91 N,. Eagleville Rd., U-3125, Storrs, CT 06269–3125., Telephone: (860) 486–4414., Fax: (860) 486–4331., E-mail:
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19
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Liu J, Li YH, Xue CF, Ding J, Gong WD, Zhao Y, Huang YX. Targeted ribonuclease can inhibit replication of hepatitis B virus. World J Gastroenterol 2003; 9:295-9. [PMID: 12532452 PMCID: PMC4611332 DOI: 10.3748/wjg.v9.i2.295] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the effect of a novel targeted ribonuclease (TN), the fusion protein of HBVc and human eosinophil-derived neurotoxin (hEDN), on the HBV replication in vitro.
METHODS: The gene encoding the targeted ribonuclease was cloned into pcDNA3.1 (-) to form recombinant eukaryotic expression vector p/TN. Control plasmids, including p/hEDN, p/HBVc, and p/TNmut in which a Lys113→Arg mutation was introduced by sequential PCR to eliminate the ribonuclease activity of hEDN, were also constructed. Liposome-mediated transfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection was performed. After that, RT-PCR was used to verify the transgene expression. Morphology of the transfected cells was observed and MTT assay was performed to detect the cytotoxicity of transgene expression. Concentration of HBsAg in the supernatant of the transfected cells was measured using solid-phase radioimmunoassay.
RESULTS: Transgenes were successfully expressed in 2.2.15 cells. No obvious cytotoxic effect of transgene expression on 2.2.15 cells was found. The HBsAg concentration in the p/TN transfected cells was reduced by 58% compared with that of mock transfected cells. No such an effect was found in all other controls.
CONCLUSION: The targeted ribonuclease can inhibit HBV replication in vitro while it has no cytotoxicity on host cells. The targeted ribonuclease may be used as a novel antiviral agent for human HBV infection.
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Affiliation(s)
- Jun Liu
- Department of Pathogenic Organism, Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China.
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20
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Abstract
There are two kinds of conformational forms of adenylate kinase (AK) in equilibrium in solution with different ANS-binding properties. Furthermore, the nature of AP(5)A inhibition suggests also that the native forms of AK for binding with different substrates pre-exist in the absence of substrates. In the present study, a kinetics approach was used to explore the native forms distinguished by ANS-binding properties and by the nature of AP(5)A inhibition. The results revealed that the native forms distinguished by ANS probe are two conformational sub-ensembles. Both sub-ensembles are active and consist of a series of forms, which pre-exist in solution and can bind with different substrates. The K(m) values of N(1) for AMP, ADP and MgATP are larger than that of N(2), indicating that the N(2) sub-ensemble is more specific for binding substrates. This is consistent with the previous observation that the activity of N(2) is about 1.8-fold of that of N(1).
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Affiliation(s)
- Yang Han
- National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, PR China
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21
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Libson AM, Gittis AG, Lattman EE. Crystal Structures of the Binary Ca2+ and pdTp Complexes and the Ternary Complex of the Asp21 .fwdarw. Glu Mutant of Staphylococcal Nuclease. Implications for Catalysis and Ligand Binding. Biochemistry 2002. [DOI: 10.1021/bi00192a004] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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22
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Abstract
Sugar non-specific endonucleases are multifunctional enzymes and are widespread in distribution. Apart from nutrition, they have also been implicated in cellular functions like replication, recombination and repair. Their ability to recognize different DNA structures has also been exploited for the determination of nucleic acid structure. Although more than 30 non-specific endonucleases have been isolated to date, very little information is available regarding their structure-function correlations except that of staphylococcal and Serratia nucleases. However, during the past few years, the primary structure, nature of the active site based on sequence homology, and the probable mechanism of action have been postulated for some of the enzymes. This review describes the purification, characteristics, biological role and applications of sugar non-specific endonucleases.
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Affiliation(s)
- E S Rangarajan
- Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India
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23
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24
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Abstract
There are two forms of rabbit muscle adenylate kinase (AK) with different 8-anilino-1-naphthalenesulfonic acid (ANS) binding properties in equilibrium solution. One form (about 70%, denoted N1) binds rapidly with ANS, whereas the other (about 30%, denoted N2) does not. Furthermore, native forms of AK should adopt different conformations for binding with substrates and products, which should be pre-existing for performing its catalytic function. The present experiments demonstrate both forms of AK distinguished by ANS probe are active. The activity of N2 is about 0.8 fold higher than N1 and shows higher susceptibility to proteolysis by trypsin. This means that the native state of AK might be an ensemble of kinetically attainable conformers and the energy landscapes of AK folding should be rugged with more than one local minimum.
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Affiliation(s)
- X Li
- National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, PR China
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25
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Filfil R, Chalikian TV. Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease. J Mol Biol 2000; 299:827-42. [PMID: 10835287 DOI: 10.1006/jmbi.2000.3773] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Deltav, accompanying the acid-induced denaturation of SNase increases from -0.013 cm(3) g(-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k(o)(s), of the protein decreases upon its unfolding with the values of Deltak(o)(s) equal to -6.3x10(-6) (-0.106 cm(3) mol(-1) bar(-1)), -4.5x10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6x10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8x10(-6) (-0.064 cm(3) mol(-1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80 % of that expected for the fully extended conformation.
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Affiliation(s)
- R Filfil
- Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, M5S 2S2, Canada
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26
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VanBrocklin M, Federspiel MJ. Capsid-targeted viral inactivation can eliminate the production of infectious murine leukemia virus in vitro. Virology 2000; 267:111-23. [PMID: 10648188 DOI: 10.1006/viro.1999.0113] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Capsid-targeted viral inactivation (CTVI), a promising gene-based antiviral strategy against retroviruses, was designed to disrupt the retroviral life cycle by incorporating a degradative enzyme (e.g., nuclease) into viral particles during assembly, thereby reducing or eliminating the production of infectious virus. The experimental system used to develop the CTVI strategy for retroviruses is designed to block the production of infectious Moloney murine leukemia virus (Mo-MLV). Two nucleases, Escherichia coli ribonulease HI and Staphylococcus nuclease, have been shown to be tolerated by the cell as Mo-MLV Gag-nuclease fusion polyproteins and still be active in the viral particles. The goal of this study was to determine what cellular and viral factors limit CTVI in cultured cells. The avian DF-1 cell line greatly expanded our ability to test the antiviral efficacy of CTVI in long-term assays and to determine the mechanism(s) of CTVI action. The CTVI antiviral effect is dependent on the level of Mo-MLV Gag-nuclease fusion polyprotein expressed. The Mo-MLV Gag-nuclease polyproteins produce a long-term prophylactic antiviral effect after a low- or high-dose Mo-MLV challenge. The Mo-MLV Gag-nuclease fusions have a significant therapeutic effect ( approximately 1000-fold) on the production of infectious Mo-MLV. The therapeutic CTVI effect can be improved by a second delivery of the CTVI fusion gene. Both the prophylactic and the therapeutic CTVI antiviral approaches can virtually eliminate the production of infectious Mo-MLV in vitro and are only limited by the number of cells in the population that do not express adequate levels of the CTVI fusion polyprotein.
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Affiliation(s)
- M VanBrocklin
- Molecular Medicine Program, Mayo Clinic and Mayo Foundation, Rochester, Minnesota, 55905, USA
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27
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Alexandrescu AT, Lamour FP, Jaravine VA. NMR evidence for progressive stabilization of native-like structure upon aggregation of acid-denatured LysN. J Mol Biol 2000; 295:239-55. [PMID: 10623523 DOI: 10.1006/jmbi.1999.3354] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The acid-denatured form of the protein LysN aggregates reversibly at pH 2.0. The strength of self-association increases with increasing Cl(-) anion concentration. At low concentrations of protein or Cl(-) anion, resonances of denatured LysN are in slow exchange with a minor form of the protein, which shows native-like NMR chemical shifts. The minor native-like resonances increase in intensity with increasing protein concentration, demonstrating that a native-like monomer fold is stabilized on aggregation of the acid-denatured protein. At high concentrations of protein or Cl(-) anion, interconversion between the major and minor resonances appears to shift from slow to intermediate exchange on the NMR timescale. NMR line-broadening is more pronounced for the major resonances of the denatured protein, which show sigmoidal decay curves with increasing Cl(-) concentration. The mid-points of the decay curves for residues in different parts of the molecule are non-coincident. We propose that differences in the NMR line-broadening transitions of individual residues reflect a stepwise stabilization of native-like structure on aggregation, starting with the segments of the protein that form the initial association interface. The resonances of the denatured protein with the greatest sensitivity to self-association correspond roughly to those that are most perturbed in the native protein on binding of the natural substrate tRNA(Lys). This suggests that the hydrophobic surfaces that promote intermolecular misfolding of acid-denatured LysN, may resemble those used for substrate binding by the native protein.
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Affiliation(s)
- A T Alexandrescu
- Department of Structural Biology, Biozentrum, University of Basel, CH-4056, Switzerland.
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28
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Johnson EP, Mincer T, Schwab H, Burgin AB, Helinski DR. Plasmid RK2 ParB protein: purification and nuclease properties. J Bacteriol 1999; 181:6010-8. [PMID: 10498713 PMCID: PMC103628 DOI: 10.1128/jb.181.19.6010-6018.1999] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The parCBA operon of the 3.2-kb stabilization region of plasmid RK2 encodes three cotranslated proteins. ParA mediates site-specific recombination to resolve plasmid multimers, ParB has been shown to be a nuclease, and the function of ParC is unknown. In this study ParB was overexpressed by cotranslation with ParC in Escherichia coli by using a plasmid construct that contained the parC and parB genes under the control of the T7 promoter. Purification was achieved by treatment of extracts with Polymin P, followed by ammonium sulfate precipitation and heparin and ion-exchange chromatography. Sizing-column analysis indicated that ParB exists as a monomer in solution. Analysis of the enzymatic properties of purified ParB indicated that the protein preferentially cleaves single-stranded DNA. ParB also nicks supercoiled plasmid DNA preferably at sites with potential single-stranded character, like AT-rich regions and sequences that can form cruciform structures. ParB also exhibits 5'-->3' exonuclease activity. This ParB activity on a 5'-end-labeled, double-stranded DNA substrate produces a 3', 5'-phosphorylated dinucleotide which is further cleaved to a 3', 5'-phosphorylated mononucleotide. The role of the ParB endonuclease and exonuclease activities in plasmid RK2 stabilization remains to be determined.
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Affiliation(s)
- E P Johnson
- Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0322, USA
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29
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Abstract
An iso-random Bi Bi mechanism has been proposed for adenylate kinase. In this mechanism, one of the enzyme forms can bind the substrates MgATP and AMP, whereas the other form can bind the products MgADP and ADP. In a catalytic cycle, the conformational changes of the free enzyme and the ternary complexes are the rate-limiting steps. The AP(5)A inhibition equations derived from this mechanism show theoretically that AP(5)A acts as a competitive inhibitor for the forward reaction and a mixed noncompetitive inhibitor for the backward reaction.
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Affiliation(s)
- X R Sheng
- National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, 100101, China
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30
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Maki K, Ikura T, Hayano T, Takahashi N, Kuwajima K. Effects of proline mutations on the folding of staphylococcal nuclease. Biochemistry 1999; 38:2213-23. [PMID: 10026306 DOI: 10.1021/bi981962+] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.
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Affiliation(s)
- K Maki
- Department of Physics, School of Science, University of Tokyo, Japan
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31
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Pedersen H, Hölder S, Sutherlin DP, Schwitter U, King DS, Schultz PG. A method for directed evolution and functional cloning of enzymes. Proc Natl Acad Sci U S A 1998; 95:10523-8. [PMID: 9724736 PMCID: PMC27927 DOI: 10.1073/pnas.95.18.10523] [Citation(s) in RCA: 95] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
A general scheme is described for the in vitro evolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.
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Affiliation(s)
- H Pedersen
- Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA
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32
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Chatfield DC, Szabo A, Brooks BR. Molecular Dynamics of Staphylococcal Nuclease: Comparison of Simulation with 15N and 13C NMR Relaxation Data. J Am Chem Soc 1998. [DOI: 10.1021/ja972215n] [Citation(s) in RCA: 108] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- David C. Chatfield
- Contribution from the Chemistry Department, Florida International University, Miami, Florida 33199, and Laboratory of Structural Biology, Division of Computer Research and Technology, and Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
| | - Attila Szabo
- Contribution from the Chemistry Department, Florida International University, Miami, Florida 33199, and Laboratory of Structural Biology, Division of Computer Research and Technology, and Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
| | - Bernard R. Brooks
- Contribution from the Chemistry Department, Florida International University, Miami, Florida 33199, and Laboratory of Structural Biology, Division of Computer Research and Technology, and Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
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33
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Zhang HJ, Sheng XR, Niu WD, Pan XM, Zhou JM. Evidence for at least two native forms of rabbit muscle adenylate kinase in equilibrium in aqueous solution. J Biol Chem 1998; 273:7448-56. [PMID: 9516443 DOI: 10.1074/jbc.273.13.7448] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The time course of 8-anilino-1-naphthalenesulfonic acid (ANS) binding to adenylate kinase (AK) is a biphasic process. The burst phase ends in the dead-time of the stopped-flow apparatus (about 15 ms), whereas the slow phase completes in about 10 min. A Job's plot tests of the binding stoichiometry demonstrates that there is one ANS binding site on AK, but only about 70% of the enzyme can rapidly bind with ANS, indicating that the conformation of native AK molecules is not homogeneous. Further kinetic analysis shows that the effects of ANS and substrates concentration on the burst and slow phase fluorescence building agree well with the multiple native forms mechanism. One form (denoted N1) binds with ANS, whereas the other (denoted N2) does not. ANS binding to N1 results in a burst phase fluorescence increase, followed by the interconversion of N2 to N1, to give the slow phase ANS binding. Under urea denaturation conditions, N2 is easily perturbed by urea and unfolds completely at low denaturant concentrations, whereas N1 is relatively resistant to denaturation and unfolds at higher denaturant concentrations. The existence of multiple native forms in solution may shed some light on the interpretation of the enzyme catalytic mechanism.
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Affiliation(s)
- H J Zhang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, China
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34
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Meeker AK, Sack GH. A fusion protein between serum amyloid A and staphylococcal nuclease—synthesis, purification, and structural studies. Proteins 1998. [DOI: 10.1002/(sici)1097-0134(19980301)30:4<381::aid-prot5>3.0.co;2-m] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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35
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Veeraraghavan S, Nall BT, Fink AL. Effect of prolyl isomerase on the folding reactions of staphylococcal nuclease. Biochemistry 1997; 36:15134-9. [PMID: 9398241 DOI: 10.1021/bi971357r] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
The low-temperature fluorescence-detected refolding of staphylococcal nuclease (SNase) can be described by three slow kinetic phases. The slowest phase is absent in the P117G mutant of SNase. Peptidyl prolyl cis-trans isomerase (cyclophilin), which has been shown to catalyze the slow folding reactions of some proteins, was employed to determine which of the refolding reactions of SNase and P117G SNase involve proline isomerization. We report here that all three folding phases of the wild type and the slower phase of P117G SNase are catalyzed by prolyl isomerase, indicating that proline isomerization is involved in these fluorescence-detected phases in the refolding of SNase. Since the rates of these phases are denaturant-dependent, we conclude that the slow folding steps involve isomerization of non-native cis proline peptide bonds and are tightly coupled to denaturant-sensitive structural changes.
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Affiliation(s)
- S Veeraraghavan
- Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760, USA
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36
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Wall ME, Ealick SE, Gruner SM. Three-dimensional diffuse x-ray scattering from crystals of Staphylococcal nuclease. Proc Natl Acad Sci U S A 1997; 94:6180-4. [PMID: 9177191 PMCID: PMC21023 DOI: 10.1073/pnas.94.12.6180] [Citation(s) in RCA: 52] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
We have developed methods for obtaining and characterizing three-dimensional maps of the reciprocal-space distribution of diffuse x-ray scattering from protein crystals, and have used the methods to study the nature of disorder in crystals of Staphylococcal nuclease. Experimentally obtained maps are 99.5% complete in the reciprocal-space resolution range of 10 A-2.5 A, show symmetry consistent with the P41 space group of the unit cell, and are highly reproducible. Quantitative comparisons of the data with three-dimensional simulations imply liquid-like motions of the protein [Caspar, D. L. D., Clarage, J., Salunke, D. M. & Clarage, M. (1988) Nature (London) 332, 659-662], with a correlation length of 10 A and a root-mean-square displacement of 0.36 A.
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Affiliation(s)
- M E Wall
- Department of Physics, Princeton University, Princeton, NJ 08544, USA
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37
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Ikura T, Tsurupa GP, Kuwajima K. Kinetic folding and cis/trans prolyl isomerization of staphylococcal nuclease. A study by stopped-flow absorption, stopped-flow circular dichroism, and molecular dynamics simulations. Biochemistry 1997; 36:6529-38. [PMID: 9174370 DOI: 10.1021/bi963174v] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
We studied the urea-induced unfolding transition of staphylococcal nuclease (SNase) and its five proline mutants (P47A, P47T, P117G, P47T/P117G, and P47A/P117G) [corrected] by peptide and aromatic circular dichroism and aromatic absorption spectroscopy at equilibrium and the refolding-unfolding kinetics of the proteins by stopped-flow circular dichroism and stopped-flow absorption techniques. Recent studies have revealed that the cis/trans isomerizations about the Pro47 and Pro117 peptide bonds of SNase occur not only in the unfolded state but also in the native state. The mutational effects on the stability and the refolding-unfolding kinetics of SNase were, however, remarkably different between the two sites. The substitution of Ala or Thr for Pro47 neither changed the stability nor affected the refolding-unfolding kinetics of SNase, whereas the substitution of Gly for Pro117 increased the protein stability by 1.2 kcal/mol (pH 7.0 and 20 degrees C) and affected the kinetics. These results have been attributed to the high flexibility of the loop around Pro47, which has been revealed by molecular dynamics simulations of native SNase. Under every condition studied, cooperative refolding-unfolding kinetics of SNase were observed. Refolding of wild-type SNase was represented by two urea concentration-dependent fast phases and a urea concentration-independent slow phase. The double mutant (P47T/P117G) [corrected] of SNase still showed multiphasic refolding kinetics that involved two urea concentration-independent slow phases, suggesting that isomerization of proline residues other than Pro47 and Pro117 may occur in the unfolded state of the mutant. Two phases were observed in the unfolding of the wild-type and mutant proteins that contained Pro117, a fast phase corresponding to the unfolding of the trans isomer and a slow phase corresponding to that of the cis isomer. On the basis of these results, the folding scheme of SNase is discussed.
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Affiliation(s)
- T Ikura
- Department of Physics, School of Science, University of Tokyo, Japan
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38
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Abstract
Staphylococcus aureus nuclease (SNase) homologues, previously thought to be restricted to bacteria and archaea, are demonstrated by sequence analysis to be present also in eukaryotes. The human cellular coactivator p100 is shown to contain four repeats, each of which is a SNase homologue. Surprisingly, these repeats are unlikely to possess SNase-like activities as each lacks equivalent SNase catalytic residues, yet they may mediate p100's single-stranded DNA-binding function. Products of Corydalis sempervirens and Saccharomyces cerevisiae open reading frames are predicted to adopt the same fold and possess similar functions as SNase. Five additional hypothetical proteins of bacterial origin are also predicted to be active SNase-like nucleases, including one that appears to be C-terminally truncated in a manner analogous to an engineered active SNase variant. Conservation of Asp-19 and Asp-83 among these homologues suggests a re-evaluation of the roles of these residues in Ca(2+)-binding and/or catalysis.
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Affiliation(s)
- C P Ponting
- Fibrinolysis Research Unit, University of Oxford, United Kingdom.
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39
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Kalnin NN, Kuwajima K. Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped-flow circular dichroism. Proteins 1995; 23:163-76. [PMID: 8592698 DOI: 10.1002/prot.340230206] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Kinetics of refolding and unfolding of staphylococcal nuclease and its six mutants, each carrying single or double amino acid substitutions, are studied by stopped-flow circular dichroism measurements. A transient kinetic intermediate formed within 10 ms after refolding starts possesses a substantial part of the N-domain core beta-structure, whereas helices are formed at the later stages. The structure of the kinetic intermediate is less organized than the structure that is known to be formed by a nuclease 1-136 fragment. Only the refolding kinetics are affected by the mutations in all the mutants except two in which the mutations have changed the native structure. From this result and also from the locations of the mutation sites, the major N-terminal domain of the nuclease in the transition state of folding has a structure nearly identical to the native one. On the other hand, the minor C-terminal domain has previously been shown to be still disorganized in the transition state. The effects of the amino acid substitutions on the stability of the native and the transition states are in good agreement with the changes in the hydration free energy, expected for the corresponding amino acid replacements in the unfolded polypeptide. Since side chains of all the mutated residues are not accessible to solvent in the native structure, the result suggests that it is the unfolded state that is mainly affected by the mutations.
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Affiliation(s)
- N N Kalnin
- Department of Physics, School of Science, University of Tokyo, Japan
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40
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Abstract
Multiple cycles of mutagenesis and phage display selection have been investigated as a method for obtaining enzymes with altered catalytic properties. A library of staphylococcal nuclease mutants displayed on phage was created by error-prone PCR mutagenesis and selected for binding to thymidine- or guanosine-containing substrate analogs. After discarding non-binders, the binding mutants were then subjected to further mutagenesis and selection rounds. After four mutagenesis and selection cycles, the catalytic properties of some of the resulting nucleases were studied and one nuclease with nine accumulated mutations was found to have a two-fold reduction in kcat for DNA hydrolysis, but a two-fold increase in kcat/Km for hydrolysis of a thymidine containing small molecule substrate. The possibility of this technique for in vitro evolution of enzyme properties is discussed.
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Affiliation(s)
- J Light
- Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037, USA
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41
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Abstract
The electrophoretic migration behavior of 12 S. nuclease variants from Staphylococcus aureus with small but well defined structural differences from site directed mutation was investigated in free solution capillary electrophoresis at pH 2.8 to 9.5. The nucleases are basic proteins; the pI and the M(r) of the wild type are 10.3 and 16.811 kd, respectively. With specially selected oligoamino buffers and with an inert, hydrophilic wall coating in 75 microns I.D. quartz capillary tubes, most of the proteins could be separated by CZE without interference by wall adsorption even at pH 9.5 where the selectivity was the highest. At pH 2.8, 4.1 and 7.0, S. nucleases are known to be in the random coil, "swollen" and the tight native state. Assuming that in a given state, i.e., at a certain pH, the molecular radii of the nucleases are the same, their hydrodynamic radii were calculated from their pertinent electrophoretic mobilities. The respective radii of 50.1, 26.8, and 25.0 Angstrum thus obtained agreed very well with the corresponding radii of gyration obtained from X-ray scattering. In fact, from the electrophoretic mobilities at pH 9.5, the existence of a hitherto unknown swollen basic state of the nuclease having a hydrodynamic radius of 30.5 Angstrum was postulated. In addition, a method was described to evaluate the valence of the protein at different pH from their pertinent electrophoretic mobilities. A general advantage of this method is that only the differences between the valences of the mutants and the wild type are needed; and for none of the proteins is required the knowledge of the actual valence. The results of the methods allowed the construction of a pH profile of the protein's valence. For the wild type, this profile was compared to the H+ titration curve and the agreement was excellent. Both methods employed some novel structure-electrophoretic mobility relationships and the predicted protein properties compared remarkably well to the values obtained by exoelectrophoretic methods such as pH titration and X-ray scattering. Surprisingly, certain S. nucleases having the same valence could also be readily separated by CZE in some cases under the same conditions used for the others. Close examination of appropriate X-ray crystallography and/or NMR data indicated subtle differences in the molecular structure of these proteins that could be responsible for slight alteration in their hydrodynamic radii.(ABSTRACT TRUNCATED AT 400 WORDS)
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Affiliation(s)
- F Kálmán
- Department of Chemical Engineering, Yale University, New Haven, CT 06520, USA
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42
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Kálmán F, Ma S, Hodel A, Fox RO, Horváth C. Charge and size effects in the capillary zone electrophoresis of nuclease A and its variants. Electrophoresis 1995; 16:595-603. [PMID: 7588531 DOI: 10.1002/elps.1150160196] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
The migration behavior of nuclease A from Staphylococcus aureus and 11 of its variants in capillary zone electrophoresis (CZE) was investigated in the light of their three-dimensional structure known from X-ray crystallography and nuclear magnetic resonance (NMR) measurements. Nuclease A (molecular mass 16.8 kDa, pKa 10.3) and the variants differ only in a single amino acid residue and have a very similar crystal structure. With the use of coated quartz capillaries and suitable buffers, the protein migration was investigated at pH from 2.8 to 9.5 without interference by wall adsorption. Although the selectivity of the electrophoretic system for the proteins was mainly determined by their charge differences, certain variants having the same net charge could also be readily separated under nondenaturing conditions. For instance, the mobility of variant K116A was sufficiently higher than that of K116G so that they could be separated by CZE. The structures of both variants are the same except for the solvent-exposed loop containing residue 116. For this reason, the difference in electrophoretic mobilities can be attributed to the fact that in K116G the backbone of the 112 to 117 amino acids protrudes slightly from the protein, with a concomitant increase in the hydrodynamic radius with respect to that of K116A. Consequently, K116G shows a smaller mobility than K116A due to its larger hydrodynamic radius despite its smaller molecular mass. The interpretation of the experimentally measured mobilities of such closely related proteins therefore requires not only consideration of their electrostatic charge but also the fine details of their molecular structures.
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Affiliation(s)
- F Kálmán
- Yale University, Department of Chemical Engineering, New Haven, CT 06520-8286, USA
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43
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Brakstad OG, Maeland JA. Direct identification of Staphylococcus aureus in blood cultures by detection of the gene encoding the thermostable nuclease or the gene product. APMIS 1995; 103:209-18. [PMID: 7755977 DOI: 10.1111/j.1699-0463.1995.tb01097.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
This study compares methods for direct identification of S. aureus in blood cultures by detection of the thermonuclease (TNase) of this bacterium or the nuc gene encoding it. The protein was detected by an enzyme diffusion test in o-toluidine blue DNA agar with a test time of at least 4 h, by a monoclonal antibody (MAb)-based sandwich enzyme-linked immunosorbent assay (sELISA) with a test time of approximately 4 h, and by a MAb-based sandwich enzyme-linked immunofiltration assay (sELIFA) with a test time of 25-30 min (sample preparation included). The nuc gene was amplified by a polymerase chain reaction (PCR) with a test time (amplification plus detection) of approximately 3.5 h. The tests were optimized for direct examination of blood-containing cultures. All tests were positive with 67/67 blood cultures which grew S. aureus, negative with 35/35 cultures which grew coagulase-negative staphylococci, and negative with 37/37 cultures with various other bacteria. These results showed positive agreement with those of the commercial AccuProbe test but not with the StaphAurex agglutination kit. With an artificially seeded blood culture, minimum total times required (incubation plus testing) were as follows: nuc-PCR, 9.5 h; sELIFA, 12.5 h; enzymatic test, 16-36 h; AccuProbe, 14 h. Direct examination of both the nuc gene and the mecA gene encoding methicillin resistance demonstrated the mecA gene in all the coagulase-negative staphylococci (48.6%) which showed oxacillin resistance. The sELIFA had the particular advantage of its short test time, the PCR its high sensitivity and the possibility of simultaneous detection of the species-specific nuc gene and genes encoding other clinically important characters of the bacteria. These tests offer the prospect of direct application to a variety of clinical specimens for rapid diagnosis of S. aureus infection.
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Affiliation(s)
- O G Brakstad
- Applied Chemistry Division, SINTEF, Trondheim, Norway
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44
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Tucker P, Edge M, Jones G, Moffatt J. The binding of dinucleoside phosphonates in the active site of the metallo-enzyme, Staphylococcal nuclease: synthetic, solution and preliminary crystallographic studies. Inorganica Chim Acta 1995. [DOI: 10.1016/0020-1693(94)04276-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
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45
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Abstract
Orotic acid is decarboxylated with a half-time (t1/2) of 78 million years in neutral aqueous solution at room temperature, as indicated by reactions in quartz tubes at elevated temperatures. Spontaneous hydrolysis of phosphodiester bonds, such as those present in the backbone of DNA, proceeds even more slowly at high temperatures, but the heat of activation is less positive, so that dimethyl phosphate is hydrolyzed with a t1/2 of 130,000 years in neutral solution at room temperature. These values extend the known range of spontaneous rate constants for reactions that are also susceptible to catalysis by enzymes to more than 14 orders of magnitude. Values of the second-order rate constant kcat/Km for the corresponding enzyme reactions are confined to a range of only 600-fold, in contrast. Orotidine 5'-phosphate decarboxylase, an extremely proficient enzyme, enhances the rate of reaction by a factor of 10(17) and is estimated to bind the altered substrate in the transition state with a dissociation constant of less than 5 x 10(-24) M.
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Affiliation(s)
- A Radzicka
- Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599
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46
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Shortle D. Staphylococcal nuclease: a showcase of m-value effects. ADVANCES IN PROTEIN CHEMISTRY 1995; 46:217-47. [PMID: 7771319 DOI: 10.1016/s0065-3233(08)60336-8] [Citation(s) in RCA: 124] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Affiliation(s)
- D Shortle
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
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47
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Deiters JA, Gallucci JC, Holmes RR. COMPUTER SIMULATION OF STAPHYLOCOCCAL NUCLEASE ACTION ON THYMIDINE 3′,5′-BIS(PHOSPHATE) (pdTp). PHOSPHORUS SULFUR 1995. [DOI: 10.1080/10426509508036958] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Affiliation(s)
- Joan A. Deiters
- a Contribution from the Department of Chemistry , University of Massachusetts , Amherst , Massachusetts , 01003
| | - Judtih C. Gallucci
- a Contribution from the Department of Chemistry , University of Massachusetts , Amherst , Massachusetts , 01003
| | - Robert R. Holmes
- a Contribution from the Department of Chemistry , University of Massachusetts , Amherst , Massachusetts , 01003
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48
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Gamble TR, Clauser KR, Kossiakoff AA. The production and X-ray structure determination of perdeuterated Staphylococcal nuclease. Biophys Chem 1994; 53:15-25. [PMID: 7841330 DOI: 10.1016/0301-4622(94)00072-7] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Staphylococcal Nuclease (SNase) has been chosen as a model protein system to evaluate the improvement in neutron diffraction data quality using fully perdeuterated protein. Large quantities of the protein were expressed in Escherichia coli grown in medium containing deuterated amino acids and deuterated water (D2O) and then purified. The mean perdeuteration level of the non-exchangable sites in the protein was found to be 96% by electrospray ionization mass spectrometry. The perdeuterated enzyme was crystallized and its X-ray structure determined. Crystals of perdeuterated SNase have been grown to 1.5 mm3. Crystallization conditions, space group and cell parameters were found to be the same for both native and perdeuterated forms of the protein. Comparison of these two forms of SNase revealed no significant structural differences between them at the atomic resolution of 1.9 A. Data collection using crystals of the perdeuterated protein is scheduled at the Brookhaven High Flux Beam Reactor.
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Affiliation(s)
- T R Gamble
- Graduate Group in Biophysics, University of California, San Francisco 94143-0448
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49
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Weber DJ, Libson AM, Gittis AG, Lebowitz MS, Mildvan AS. NMR docking of a substrate into the X-ray structure of the Asp-21-->Glu mutant of staphylococcal nuclease. Biochemistry 1994; 33:8017-28. [PMID: 8025106 DOI: 10.1021/bi00192a005] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
To understand the structural basis of the 1500-fold decrease in catalytic activity of the D21E mutant of staphylococcal nuclease in which an aspartate ligand of the essential Ca2+ has been enlarged to glutamate, the conformation of the enzyme-bound substrate dTdA has been determined by NMR methods and has been docked into the X-ray structure of the D21E mutant (Libson, A. M., Gittis, A.G., & Lattman, E. E. Biochemistry, preceding paper in this issue) based on distances from the bound metal ion to dTdA and on intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of dTdA, using energy minimization to relieve small overlaps. Like the wild-type enzyme, the D21E mutant forms binary E-M and E-S and ternary E-M-S complexes with Ca2+, Mn2+, Co2+, and La3+. D21E enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 of the phosphorus and on 1/T1 of four proton resonances of dTdA, and these effects are abolished by the binding of the competitive inhibitor 3',5'-pdTp. From the paramagnetic effects of enzyme-bound Co2+ on 1/T1 of phosphorus and protons, with the use of a correlation time of 1.1 ps based on 1/T1 values at 250 and 600 MHz, five metal-nucleus distances and 11 lower limit metal-nucleus distances have been calculated. The Co2+ to 31P distance of 4.1 +/- 0.9 A agrees with that found on the wild-type enzyme (Weber, D. J., Mullen, G. P., & Mildvan, A. S. (1991) Biochemistry 30, 7425-7437) and indicates at least 18% inner sphere phosphate coordination. Fourteen interproton distances and 109 lower limit interproton distances in dTdA in the ternary D21E-La(3+)-dTdA complex were determined by NOESY spectra at 50-, 100-, and 200-ms mixing times. Both the metal-nucleus and interproton distances were necessary to compute a narrow range of conformations for enzyme-bound dTdA. As on the wild-type enzyme, the conformation of dTdA on the D21E mutant is highly extended, with high-anti C-2' endo conformations for the individual nucleosides. However, significant conformational differences are found in the torsional angles chi of dA (delta chi = 49 +/- 3 degrees), in gamma of dT (delta gamma = 108 +/- 30 degrees) and in zeta of dT (delta zeta = 124 +/- 38 degrees).(ABSTRACT TRUNCATED AT 400 WORDS)
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Affiliation(s)
- D J Weber
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185
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50
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Chuang WJ, Gittis AG, Mildvan AS. Magnetic resonance studies of the binding of oligonucleotide substrates to mutants of staphylococcal nuclease. Proteins 1994; 18:68-80. [PMID: 8146123 DOI: 10.1002/prot.340180109] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
By a combination of NMR docking and model building, the substrate binding site on staphylococcal nuclease was found to accommodate a trinucleotide and to consist of three subsites, each interacting with a single nucleotidyl unit of DNA. Binding of the essential Ca2+ activator and substrate cleavage occur between subsites 1 and 2. Hence, catalytically productive binding would span subsites 1 and 2 while nonproductive binding would span subsites 2 and 3. Lys-49 is near subsite 1, and Lys-84 and Tyr-115 interact with substrates at subsite 3 [Weber, D.J., Gittis, A.G., Mullen, G.P., Abeygunawardana, C., Lattman, E.E., Mildvan, A.S. Proteins 13:275-287, 1992]. The proposed locations of these subsites were independently tested by the effects of the K49A, K84A, and Y115A mutations of staphylococcal nuclease on the binding of Mn2+, Ca2+, and the dinucleotide and trinucleotide substrates, 5'-pdTdA, dTdA, and dTdAdG. These three mutants have previously been shown to be fully active and to have CD and 2D NMR spectra very similar to those of the wild-type enzyme (Chuang, W.-J., Weber, D.J., Gittis, A.G., Mildvan, A.S. Proteins 17:36-48, 1993). All three mutant enzymes and their pdTdA and dTdA complexes (but not their dTdAdG complex) bind Mn2+ and Ca2+ more weakly than the wild-type enzyme by factors ranging from 2 to 11. The presence of a terminal phosphate as in 5'-pdTdA raises the affinity of the substrate for staphylococcal nuclease and its three mutants by two orders of magnitude and for the corresponding enzyme-metal complexes by three to four orders of magnitude, suggesting that the terminal phosphate is coordinated by the enzyme-bound divalent cation. Such complexation would result in the nonproductive binding of 5'-pdTdA at subsites 2 and 3. Accordingly, the K84A and Y115A mutations significantly weaken the binding of 5'-pdTdA and its metal to staphylococcal nuclease by factors of 2.2 to 37.8, while the K49A mutation has much smaller or no effect. Such nonproductive binding explains the low activity of staphylococcal nuclease with small substrates, especially those with a terminal phosphate. Similarly, the K84A and Y115A mutations weaken the binding of dTdA and its metal complexes to the enzyme by factors of 3.4 to 13.1 while the K49A mutation has smaller effects indicating significant nonproductive binding of dTdA. The trinucleotide dTdAdG binds more tightly to wild-type and mutant staphylococcal nuclease and to its metal complexes than does the dinucleotide dTdA by factors of 2.4 to 12.2, reflecting the occupancy of an additional subsite.(ABSTRACT TRUNCATED AT 400 WORDS)
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Affiliation(s)
- W J Chuang
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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