Genetically encoded, fluorescent protein (FP)-based Förster resonance energy transfer (FRET) biosensors are microscopy imaging tools tailored for the precise monitoring and detection of molecular dynamics within subcellular microenvironments. They are characterised by their ability to provide an outstanding combination of spatial and temporal resolutions in live-cell microscopy. In this review, we begin by tracing back on the historical development of genetically encoded FP labelling for detection in live cells, which lead us to the development of early biosensors and finally to the engineering of single-chain FRET-based biosensors that have become the state-of-the-art today. Ultimately, this review delves into the fundamental principles of FRET and the design strategies underpinning FRET-based biosensors, discusses their diverse applications and addresses the distinct challenges associated with their implementation. We place particular emphasis on single-chain FRET biosensors for the Rho family of guanosine triphosphate hydrolases (GTPases), pointing to their historical role in driving our understanding of the molecular dynamics of this important class of signalling proteins and revealing the intricate relationships and regulatory mechanisms that comprise Rho GTPase biology in living cells.

In cells, wild-type RasGTP complexes exist in two distinct states: active State 2 and inactive State 1. These complexes regulate their functions by transitioning between the two states. However, the mechanisms underlying this state transition have not been clearly elucidated. To address this, we conducted a detailed simulation study to characterize the energetics of the stable states involved in the state transitions of the HRasGTP complex, specifically from State 2 to State 1. This was achieved by employing multiscale quantum mechanics/molecular mechanics and enhanced sampling molecular dynamics methods. Based on the simulation results, we constructed the two-dimensional free energy landscapes that provide crucial information about the conformational changes of the HRasGTP complex from State 2 to State 1. Furthermore, we also explored the conformational changes from the intermediate state to the product state during guanosine triphosphate hydrolysis. This study on the conformational changes involved in the HRas state transitions serves as a valuable reference for understanding the corresponding events of both KRas and NRas as well.

The mechanism of the deceptively simple reaction of guanosine triphosphate (GTP) hydrolysis catalyzed by the cellular protein Ras in complex with the activating protein GAP is an important issue because of the significance of this reaction in cancer research. We show that molecular modeling of GTP hydrolysis in the Ras-GAP active site reveals a diversity of mechanisms of the intrinsic chemical reaction depending on molecular groups at position 61 in Ras occupied by glutamine in the wild-type enzyme. First, a comparison of reaction energy profiles computed at the quantum mechanics/molecular mechanics (QM/MM) level shows that an assignment of the Gln61 side chain in the wild-type Ras either to QM or to MM parts leads to different scenarios corresponding to the glutamine-assisted or the substrate-assisted mechanisms. Second, replacement of Gln61 by the nitro-analog of glutamine (NGln) or by Glu, applied in experimental studies, results in two more scenarios featuring the so-called two-water and the concerted-type mechanisms. The glutamine-assisted mechanism in the wild-type Ras-GAP, in which the conserved Gln61 plays a decisive role, switching between the amide and imide tautomer forms, is consistent with the known experimental results of structural, kinetic and spectroscopy studies. The results emphasize the role of the Ras residue Gln61 in Ras-GAP catalysis and explain the retained catalytic activity of the Ras-GAP complex towards GTP hydrolysis in the Gln61NGln and Gln61Glu mutants of Ras.

Amide-imide tautomerization presents a pervasive class of chemical transformations in organic chemistry of natural compounds. In this Perspective, we describe two distinctively different protein systems, in which the amide-imide tautomerization in the glutamine side chain takes place in enzymatic or photochemical reactions. First, hydrolysis of guanosine triphosphate (GTP) catalyzed by the Ras-GAP protein complex suggests the occurrence of the imide tautomer of glutamine in reaction intermediates. Second, photoexcitation of flavin-binding protein domains (BLUFs) initiates a chain of reactions in the chromophore-binding pocket, including amide-imide tautomerization of glutamine. Mechanisms of these reactions at the atomic level have been revealed in quantum mechanics/molecular mechanics (QM/MM) simulations. To reinforce conclusions on the critical role of amide-imide tautomerization of glutamine in these reactions we describe results of new quantum chemistry and QM/MM calculations for relevant molecular model systems. We reexamine results of the recent IR spectroscopy studies of BLUF domains, which provide experimental evidences of Gln tautomerization in proteins. We also propose to validate the glutamine-assisted mechanism of enzymatic GTP hydrolysis by using IR spectroscopy in a proper range of wavenumbers.

Our quantum chemical activation strain analyses demonstrate how Mg2+ lowers the barrier of the enzymatic triphosphate hydrolysis through two distinct mechanisms: (a) weakening of the leaving-group bond, thereby decreasing activation strain; and (b) transition state (TS) stabilization through enhanced electrophilicity of the triphosphate PPP substrate, thereby strengthening the interaction with the nucleophile.

The most representative member of the Ras subfamily is its HRas isoform. Ras proteins being GTPases, possess an intrinsic activity to hydrolyze the GTP molecule to GDP. During the transition phases, between active and inactive states, P-loop and switch regions show maximum variations. Various hot-spot Ras mutants (G12V, A59G, Q61L etc) have been reported, that limit the protein's conformation in the permanent active state. In the present study, we aim to explore the structural dynamics of one such crucial mutant of Ras namely A59G which belongs to the conserved Switch II region of the protein. Approximately ∼15μs of Classical Molecular Dynamics (CMD) simulations have been carried out on the mutant and wild-type complexes. Further, a metadynamics simulation of 500ns was also carried out, which suggests an energy barrier of ∼9.56kcal/mol between wild-type and mutant conformation. We demonstrate the role of water molecule in maintaining the required interaction networks in the pre-hydrolysis state, its impact on A59G mutation, distinct orientation of the Gln61 residue in two conformations, disruption of crucial Gly60 and γ phosphate and the change in the Switch II region. The outcome of our study captures the pre-hydrolysis state of the HRas protein. It also establishes the fact that this mutation makes the movement of Switch II region and the conserved DXXGQ motif highly constrained, which is known to be an important requirement for hydrolysis. This suggests that the A59G mutation may decrease the rate of intrinsic hydrolysis as well as GAP-mediated hydrolysis.

Despite years of study, the structural or dynamical basis for the differential reactivity and oncogenicity of Ras isoforms and mutants remains unclear. In this study, we investigated the effects of amino acid variations on the structure and dynamics of wild type and oncogenic mutants G12D, G12V, and G13D of H- and K-Ras proteins. Based on data from µs-scale molecular dynamics simulations, we show that the overall structure of the proteins remains similar but there are important differences in dynamics and interaction networks. We identified differences in residue interaction patterns around the canonical switch and distal loop regions, and persistent sodium ion binding near the GTP particularly in the G13D mutants. Our results also suggest that different Ras variants have distinct local structural features and interactions with the GTP, variations that have the potential to affect GTP release and hydrolysis. Furthermore, we found that H-Ras proteins and particularly the G12V and G13D variants are significantly more flexible than their K-Ras counterparts. Finally, while most of the simulated proteins sampled the effector-interacting state 2 conformational state, G12V and G13D H-Ras adopted an open switch state 1 conformation that is defective in effector interaction. These differences have implications for Ras GTPase activity, effector or exchange factor binding, dimerization and membrane interaction. Proteins 2017; 85:1618-1632. © 2017 Wiley Periodicals, Inc.

The phosphoryl group, PO₃^-, is the dynamic structural unit in the biological chemistry of phosphorus. Its transfer from a donor to an acceptor atom, with oxygen much more prevalent than nitrogen, carbon, or sulfur, is at the core of a great majority of enzyme-catalyzed reactions involving phosphate esters, anhydrides, amidates, and phosphorothioates. The serendipitous discovery that the phosphoryl group could be labeled by "nuclear mutation," by substitution of PO₃^- by MgF₃^- or AlF₄^-, has underpinned the application of metal fluoride (MF _x ) complexes to mimic transition states for enzymatic phosphoryl transfer reactions, with sufficient stability for experimental analysis. Protein crystallography in the solid state and ¹⁹F NMR in solution have enabled direct observation of ternary and quaternary protein complexes embracing MF _x transition state models with precision. These studies have underpinned a radically new mechanistic approach to enzyme catalysis for a huge range of phosphoryl transfer processes, as varied as kinases, phosphatases, phosphomutases, and phosphohydrolases. The results, without exception, have endorsed trigonal bipyramidal geometry (tbp) for concerted, "in-line" stereochemistry of phosphoryl transfer. QM computations have established the validity of tbp MF _x complexes as reliable models for true transition states, delivering similar bond lengths, coordination to essential metal ions, and virtually identical hydrogen bond networks. The emergence of protein control of reactant orbital overlap between bond-forming species within enzyme transition states is a new challenging theme for wider exploration.

GTPases play a crucial role in the regulation of many biological processes by catalyzing the hydrolysis of GTP into GDP. The focus of this work is on the dynamin-related large GTPase human guanine nucleotide binding protein-1 (hGBP1) which is able to hydrolyze GTP even to GMP. Here, we studied the largely unknown mechanisms of both GTP and GDP hydrolysis steps utilizing accelerated ab initio QM/MM metadynamics simulations to compute multi-dimensional free energy landscapes. We find an indirect substrate-assisted catalysis (SAC) mechanism for GTP hydrolysis involving transfer of a proton from the water nucleophile to a nonbridging phosphoryl oxygen via a proton relay pathway where the rate-determining first step is concerted-dissociative nature. A "composite base" consisting of Ser73, Glu99, a bridging water molecule, and GTP was found to activate the nucleophilic water, thus disclosing the complex nature of the general base in hGBP1. A nearly two-fold reduction in the free energy barrier was obtained for GTP hydrolysis in the enzyme in comparison to bulk solvent. The subsequent GDP hydrolysis in hGBP1 was also found to follow a water-mediated proton shuttle mechanism. It is expected that the proton shuttle mechanisms unravelled for hGBP1 apply to many classes of GTPases/ATPases that possess an optimally-arranged hydrogen bonding network, which connects the catalytic water to a proton acceptor.

The structures and vibrational spectra of the reacting species upon guanosine triphosphate (GTP) hydrolysis to guanosine diphosphate and inorganic phosphate (Pi) trapped inside the protein complex Ras-GAP were analyzed following the results of QM/MM simulations. The frequencies of the phosphate vibrations referring to the reactants and to Pi were compared to those observed in the experimental FTIR studies. A good correlation between the theoretical and experimental vibrational data provides a strong support to the reaction mechanism of GTP hydrolysis by the Ras-GAP enzyme system revealed by the recent QM/MM modeling. Evolution of the vibrational bands associated with the inorganic phosphate Pi during the elementary stages of GTP hydrolysis is predicted.

We address methodological issues in quantum mechanics/molecular mechanics (QM/MM) calculations on a zinc-dependent enzyme. We focus on the first stage of peptide bond cleavage by matrix metalloproteinase-2 (MMP-2), that is, the nucleophilic attack of the zinc-coordinating water molecule on the carbonyl carbon atom of the scissile fragment of the substrate. This step is accompanied by significant charge redistribution around the zinc cation, bond cleavage, and bond formation. We vary the size and initial geometry of the model system as well as the computational protocol to demonstrate the influence of these choices on the results obtained. We present QM/MM potential energy profiles for a set of snapshots randomly selected from QM/MM-based molecular dynamics simulations and analyze the differences in the computed profiles in structural terms. Since the substrate in MMP-2 is located on the protein surface, we investigate the influence of the thickness of the water layer around the enzyme on the QM/MM energy profile. Thin water layers (0-2 Å) give unrealistic results because of structural reorganizations in the active-site region at the protein surface. A 12 Å water layer appears to be sufficient to capture the effect of the solvent; the corresponding QM/MM energy profile is very close to that obtained from QM/MM/SMBP calculations using the solvent macromolecular boundary potential (SMBP). We apply the optimized computational protocol to explain the origin of the different catalytic activity of the Glu116Asp mutant: the energy barrier for the first step is higher, which is rationalized on structural grounds. © 2016 Wiley Periodicals, Inc.

Complexes of small GTPases with GTPase-activating proteins have been intensively studied with the main focus on the complex of H-Ras with p120GAP (Ras-GAP). The detailed mechanism of GTP hydrolysis is still unresolved. To clarify it, we calculated the energy profile of GTP hydrolysis in the active site of a recently characterized vision-related member of this family, the Arl3-RP2 complex. The mechanism suggested in this study retains the main features of GTP hydrolysis by the Ras-GAP complex, but the relative energies of the corresponding intermediates are different and an additional intermediate exists in the Arl3-RP2 complex compared with the Ras-GAP. These differences arise from small deviations in the catalytic arginine conformation of the active site. In the Arl3-RP2 complex, the first two intermediates, corresponding to the Pγ-Oβγ bond cleavage and the glutamine-assisted proton transfer, are almost isoenergetic with the ES complex. Numerical simulations of the kinetic curves demonstrate that the concentrations of these intermediates are comparable with that of ES during the reaction. The calculated IR spectra reveal specific vibrational bands, corresponding to these intermediates. These specific features of the Arl3-RP2 complex open the opportunity to identify spectroscopically two more reaction intermediates in GTP hydrolysis in addition to the ES and EP complexes.

Interpretation of the experiments showing that the Ras-GAP protein complex maintains activity in guanosine triphosphate (GTP) hydrolysis upon replacement of Glu61 in Ras with its unnatural nitro analog, NGln, is an important issue for understanding details of chemical transformations at the enzyme active site. By using molecular modeling we demonstrate that both glutamine and its nitro analog in the aci-nitro form participate in the reaction of GTP hydrolysis at the stages of proton transfer and formation of inorganic phosphate. The computed structures and the energy profiles for the complete pathway from the enzyme-substrate to enzyme-product complexes for the wild-type and mutated Ras suggest that the reaction mechanism is not affected by this mutation.

Molecular mechanisms of the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by the Ras·GAP protein complex are fully investigated by using modern modeling tools. The previously hypothesized stages of the cleavage of the phosphorus-oxygen bond in GTP and the formation of the imide form of catalytic Gln61 from Ras upon creation of Pi are confirmed by using the higher-level quantum-based calculations. The steps of the enzyme regeneration are modeled for the first time, providing a comprehensive description of the catalytic cycle. It is found that for the reaction Ras·GAP·GTP·H2O → Ras·GAP·GDP·Pi, the highest barriers correspond to the process of regeneration of the active site but not to the process of substrate cleavage. The specific shape of the energy profile is responsible for an interesting kinetic mechanism of the GTP hydrolysis. The analysis of the process using the first-passage approach and consideration of kinetic equations suggest that the overall reaction rate is a result of the balance between relatively fast transitions and low probability of states from which these transitions are taking place. Our theoretical predictions are in excellent agreement with available experimental observations on GTP hydrolysis rates.