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Nuclear magnetic resonance (NMR) has the unique advantage of elucidating the structure and dynamics of biomolecules in solution at physiological temperatures, where they are in constant movement on timescales from picoseconds to milliseconds. Such motions have been shown to be critical for enzyme catalysis, allosteric regulation, and molecular recognition. With NMR being particularly sensitive to these timescales, detailed information about the kinetics can be acquired. However, nearly all methods of NMR-based biomolecular structure determination neglect kinetics, which introduces a large approximation to the underlying physics, limiting both structural resolution and the ability to accurately determine molecular flexibility. Here we present the Kinetic Ensemble approach that uses a hierarchy of interconversion rates between a set of ensemble members to rigorously calculate Nuclear Overhauser Effect (NOE) intensities. It can be used to simultaneously refine both temporal and structural coordinates. By generalizing ideas from the extended model free approach, the method can analyze the amplitudes and kinetics of motions anywhere along the backbone or side chains. Furthermore, analysis of a large set of crystal structures suggests that NOE data contains a surprising amount of high-resolution information that is better modeled using our approach. The Kinetic Ensemble approach provides the means to unify numerous types of experiments under a single quantitative framework and more fully characterize and exploit kinetically distinct protein states. While we apply the approach here to the protein ubiquitin and cross validate it with previously derived datasets, the approach can be applied to any protein for which NOE data is available. Electronic supplementary material The online version of this article (10.1007/s10858-019-00288-8) contains supplementary material, which is available to authorized users.

The recent advances in relative protein–ligand binding free energy calculations have shown the value of alchemical methods in drug discovery. Accurately assessing absolute binding free energies, although highly desired, remains a challenging endeavour, mostly limited to small model cases. Here, we demonstrate accurate first principles based absolute binding free energy estimates for 128 pharmaceutically relevant targets. We use a novel rigorous method to generate protein–ligand ensembles for the ligand in its decoupled state. Not only do the calculations deliver accurate protein–ligand binding affinity estimates, but they also provide detailed physical insight into the structural determinants of binding. We identify subtle rotamer rearrangements between apo and holo states of a protein that are crucial for binding. When compared to relative binding free energy calculations, obtaining absolute binding free energies is considerably more challenging in large part due to the need to explicitly account for the protein in its apo state. In this work we present several approaches to obtain apo state ensembles for accurate absolute ΔG calculations, thus outlining protocols for prospective application of the methods for drug discovery., Molecular dynamics based absolute protein–ligand binding free energies can be calculated accurately and at large scale to facilitate drug discovery.

Understanding protein function requires detailed knowledge about protein dynamics , i.e. the different conformational states the system can adopt. Despite substantial experimental progress, simulation techniques such as molecular dynamics (MD) currently provide the only routine means to obtain dynamical information at an atomic level on timescales of nano- to microseconds. Even with the current development of computational power, sampling techniques beyond MD are necessary to enhance conformational sampling of large proteins and assemblies thereof. The use of collective coordinates has proven to be a promising means in this respect, either as a tool for analysis or as part of new sampling algorithms. Starting from MD simulations, several enhanced sampling algorithms for biomolecular simulations are reviewed in this chapter. Examples are given throughout illustrating how consideration of the dynamic properties of a protein sheds light on its function.

Background and purpose: Pentamidine is a drug used in treatment of protozoal infections. Pentamidine treatment may cause sudden cardiac death by provoking cardiac arrhythmias associated with QTc prolongation and U-wave alterations. This proarrhythmic effect was linked to inhibition of hERG trafficking, but not to acute block of ion channels contributing to the action potential. Because the U-wave has been linked to the cardiac inward rectifier current (IK1), we examined the action and mechanism of pentamidine-mediated IK1 block. Experimental approach: Patch clamp measurements of IK1 were made on cultured adult canine ventricular cardiomyocytes, KIR2.1-HEK293 cells and KIR2.x inside-out patches. Pentamidine binding to cytoplasmic amino acid residues of KIR2.1 channels was studied by molecular modelling. Key results: Pentamidine application (24 h) decreased IK1 in cultured canine cardiomyocytes and KIR2.1-HEK293 cells under whole cell clamp conditions. Pentamidine inhibited IK1 in KIR2.1-HEK293 cells 10 min after application. When applied to the cytoplasmic side under inside-out patch clamp conditions, pentamidine block of IK1 was acute (IC50= 0.17 µM). Molecular modelling predicted pentamidine-channel interactions in the cytoplasmic pore region of KIR2.1 at amino acids E224, D259 and E299. Mutation of these conserved residues to alanine reduced pentamidine block of IK1. Block was independent of the presence of spermine. KIR2.2, and KIR2.3 based IK1 was also sensitive to pentamidine blockade. Conclusions and implications: Pentamidine inhibits cardiac IK1 by interacting with three negatively charged amino acids in the cytoplasmic pore region. Our findings may provide new insights for development of specific IK1 blocking compounds.

SoPIP2;1 is one of the major integral proteins in spinach leaf plasma membranes. In the Xenopus oocyte expression system its water channel activity is regulated by phosphorylation at the C terminus and in the first cytosolic loop. To assess its structure, SoPIP2;1 was heterologously expressed in Pichia pastoris as a His-tagged protein and in the non-tagged form. Both forms were reconstituted into 2D crystals in the presence of lipids. Tubular crystals and double-layered crystalline sheets of non-tagged SoPIP2;1 were observed and analyzed by cryo-electron microscopy. Crystalline sheets were highly ordered and diffracted electrons to a resolution of 2.96A. High-resolution projection maps of tilted specimens provided a 3D structure at 5A resolution. Superposition of the SoPIP2;1 potential map with the atomic model of AQP1 demonstrates the generally well conserved overall structure of water channels. Differences concerning the extracellular loop A explain the particular crystal contacts between oppositely oriented membrane sheets of SoPIP2;1 2D crystals, and may have a function in rapid volume changes observed in stomatal guard cells or mesophyll protoplasts. This crystal packing arrangement provides access to the phosphorylated C terminus as well as the loop B phosphorylation site for studies of channel gating.

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two- dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to- noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at subnanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

A principal component analysis has been applied on equilibrium simulations of a b-heptapeptide that shows reversible folding in a methanol solution. The analysis shows that the configurational space contains only three dense sub-states. These states of relatively low free energy correspond to the ‘‘native’’ left-handed helix, a partly helical intermediate, and a hairpin-like structure. The collection of unfolded conformations form a relatively diffuse cloud with little substructure. Internal hydrogen-bonding energies were found to correlate well with the degree of folding. The native helical structure folds from the N terminus; the transition from the major folding intermediate to the native helical structure involves the formation of the two most C-terminal backbone hydrogen bonds. A fourstate Markov model was found to describe transition frequencies between the conformational states within error limits, indicating that memoryeffects are negligible beyond the nanosecond time-scale. The dominant native state fluctuations were found to be very similar to unfolding motions, suggesting that unfolding pathways can be inferred from fluctuations in the native state. The low-dimensional essential subspace, describing 69 % of the collective atomic fluctuations, was found to converge at time-scales of the order of one nanosecond at all temperatures investigated, whereas folding/unfolding takes place at significantly longer time-scales, even above the melting temperature. # 2001 Academic Press

The dynamics of collective protein motions derived from Molecular Dynamics simula- tions have been studied for two small model proteins: initiation factor I and the B1 domain of Protein G. First, we compared the structural fluctuations, ob- tained by local harmonic approximations in differ- ent energy minima, with the ones revealed by large scale molecular dynamics (MD) simulations. It was found that a limited set of harmonic wells can be used to approximate the configurational fluctua- tions of these proteins, although any single har- monic approximation cannot properly describe their dynamics. Subsequently, the kinetics of the main (essential) collective protein motions were characterized. A dual-diffusion behavior was observed in which a fast type of diffusion switches to a much slower type in a typical time of about 1-3 ps. From these results, the large backbone conformational fluctuations of a protein may be considered as ''hopping'' between multiple harmonic wells on a basically flat free energy surface. Proteins 1999;35:283-292.

Conformational changes are known to play a crucial role in the function of the bacterial GroE chaperonin system. Here, results are presented from an essential dynamics analysis of known experimental structures and from computer simulations of GroEL using the CONCOORD method. The results indicate a possible direct form of inter-ring communication associated with internal fluctuations in the nucleotide-binding domains upon nucleotide and GroES binding that are involved in the allosteric mechanism of GroEL. At the level of conformational transitions in entire GroEL rings, nucleotide-induced structural changes were found to be distinct and in principle uncoupled from changes occurring upon GroES binding. However, a coupling is found between nucleotide-induced conformational changes and GroES-mediated transitions, but only in simulations of GroEL double rings, and not in simulations of single rings. This provides another explanation for the fact that GroEL functions a double ring system.

A method is presented to mathematically extract concerted structural transitions in proteins from collections of crystal structures. The ''essential dynamics'' procedure is used to filter out small-amplitude fluctuations from such a set of structures; the remaining large conformational changes describe motions such as those important for the uptake/release of substrate/ligand and in catalytic reactions. The method is applied to sets of x-ray structures for a number of proteins, and the results are compared with the results from essential dynamics as applied to molecular dynamics simulations of those proteins. A significant degree of similarity is found, thereby providing a direct experimental basis for the application of such simulations to the description of large concerted motions in proteins.

Recently, we developed a method (Amadei et al., J. Biomol. Str. Dyn. 13: 615-626; de Groot et al., J. Biomol. Str. Dyn. 13: 741-751, 1996) to obtain an extended sampling of the configurational space of proteins, using an adapted form of molecular dynamics (MD) simulations, based on the essential dynamics (ED) (Amadei et al., Proteins 17:412-425, 1993) method. In the present study, this ED sampling technique is applied to the histidine-containing phosphocarrier protein HPr from Escherichia coli. We find a cluster of conformations that is an order of magnitude larger than that found for a usual MD simulation of comparable length. The structures in this cluster are geometrically and energetically comparable to NMR structures. Moreover, on average, this large cluster satisfies nearly all NMR-derived distance restraints.

A detailed investigation is presented into the effect of limited sampling time and small changes in the force field on molecular dynamics simulations of a protein. Thirteen independent simulations of the B1 IgG-binding domain of streptococcal protein G were performed, with small changes in the simulation parameters in each simulation. Parameters studied included temperature, bond constraints, cut-off radius for electrostatic interactions, and initial placement of hydrogen atoms. The essential dynamics technique was used to reveal dynamic differences between the simulations. Similar essential dynamics properties were found for all simulations, indicating that the large concerted motions found in the simulations are not particularly sensitive to small changes in the force field. A thorough investigation into the stability of the essential dynamics properties as derived from a molecular dynamics simulation of a few hundred picoseconds is provided. Although the definition of the essential modes of motion has not fully converged in these short simulations, the subspace in which these modes are confined is found to be reproducible.

The recently introduced Essential Dynamics sampling method is extended such that an exhaustive sampling of the available (backbone) configurational space can be achieved. From an initial Molecular Dynamics simulation an approximated definition of the essential subspace is obtained. This subspace is used to direct subsequent simulations by means of constraint forces. The method is applied to the peptide hormone guanylin, solvated in water, of which the structure was determined recently. The peptide exists in two forms and for both forms, an extensive sampling was produced. The sampling algorithm fills the available space (of the essential coordinates used in the procedure) at a rate that is approximately six to seven times larger than that for traditional Molecular Dynamics. The procedure does not cause any significant perturbation, which is indicated by the fact that free Molecular Dynamics simulations started at several places in the space defined by the Essential Dynamics sample that complete space. Moreover, analyses of the average free Molecular Dynamics step have shown that nowhere except close to the edge of the available space, there are regions where the system shows a drift in a particular direction. This result also shows that in principle, the essential subspace is a constant free energy surface, with well-defined and steep borders, in which the system moves diffusively. In addition, a comparison between two independent essential dynamics sampling runs, of one form of the peptide, shows that the obtained essential subspaces are virtually identical.

Cell volume and water permeability measurements in cultured mammalian cells are typically conducted under a light microscope. Many of the employed approaches are time consuming and not applicable to a study of confluent epithelial cell monolayers. We present here an adaptation of a calcein-quenching-based approach for a plate reader. A standard curve of fluorescence intensities at equilibrium has been recorded, following a shift from 285 mosmol/kgH2O to a series of altered extracellular osmolyte concentrations, ranging from final concentrations of 185 to 585 mosmol/kgH2O, by changing buffer d-mannitol concentrations. Similarly, according average cell volumes have been measured in suspension in a Coulter counter (particle-sizing device). Based on these measurements, we have derived an equation that facilitates the modeling of cell volume changes based on fluorescence intensity changes. We have utilized the method to study the role of a carboxyl-terminus aquaporin (AQP)-2 phosphorylation site, which is known to affect AQP2 membrane trafficking, in heterologous type I Madin-Darby canine kidney cells. We find that water permeability in cells expressing phosphorylation site mutants was in the following order: AQP2-S256D > AQP2 wild-type > AQP2-S256A. We propose that the method can be applied to study AQP function and more generally to study cell volume changes in adherent cell lines. Furthermore, it should be adaptable for AQP inhibitor screening in chemical compound libraries.

Normal mode analysis and essential dynamics analysis are powerful methods used for the analysis of collective motions in biomolecules. Their application has led to an appreciation of the importance of protein dynamics in function and the relationship between structure and dynamical behavior. In this chapter, the methods and their implementation are introduced and recent developments such as elastic networks and advanced sampling techniques are described.

Introduction: The UMCG is the only medical centre in the Netherlands that has performed liver transplants on children since 1982. In the last few years a lot has improved in the treatment thereof. At this current moment approximately 15 children are being transplanted each year. The survival rate has improved by 50%. During these admissions pitfalls may occur that can lead to postoperative complications! Aim: To give insight on the means used to recognize post operative complications in early stages in children after liver transplants. Methods: Protocols have been developed in which post operative parameters concerning monitoring, blood results and radiology imaging have been described. Nurse observation and interpretation parameters are of further essential significance. These are mainly directed towards prevention of infections, timely detubation, and keeping up with the fluid balance and temperature of the patient. Adjacent to that, the in research phase TEG-study, offers important information about the coagulation. Results: Working according to protocol makes it possible to perceive and intercept complications in early stages. Moreover we aim at thrombosis, infections and liver failure. In pediatric care parents are indissolubly attached to their child. This makes parental involvement in children's admissions of essential value. Conclusion: We use protocols that can help detect complications in an early stage after liver transplants.

The dynamics of collective protein motions derived from Molecular Dynamics simulations have been studied for two small model proteins: initiation factor I and the B1 domain of Protein G. First, we compared the structural fluctuations, obtained by local harmonic approximations in different energy minima, with the ones revealed by large scale molecular dynamics (MD) simulations. It was found that a limited set of harmonic wells can be used to approximate the configurational fluctuations of these proteins, although any single harmonic approximation cannot properly describe their dynamics. Subsequently, the kinetics of the main (essential) collective protein motions were characterized. A dual-diffusion behavior was observed in which a fast type of diffusion switches to a much slower type in a typical time of about 1-3 ps. From these results, the large backbone conformational fluctuations of a protein may be considered as "hopping" between multiple harmonic wells on a basically flat free energy surface.

Conformational changes are known to play a crucial role in the function of the bacterial GroE chaperonin system. Here, results are presented from an essential dynamics analysis of known experimental structures and from computer simulations of GroEL using the CONCOORD method. The results indicate a possible direct form of inter-ring communication associated with internal fluctuations in the nucleotide-binding domains upon nucleotide and GroES binding that are involved in the allosteric mechanism of GroEL. At the level of conformational transitions in entire GroEL rings, nucleotide-induced structural changes were found to be distinct and in principle uncoupled from changes occurring upon GroES binding. However, a coupling is found between nucleotide-induced conformational changes and GroES-mediated transitions, but only in simulations of GroEL double rings, and not in simulations of single rings. This provides another explanation for the fact that GroEL functions a double ring system.

A comparison of a series of extended molecular dynamics (MD) simulations of bacteriophage T4 lysozyme in solvent with X-ray data is presented. Essential dynamics analyses were used to derive collective fluctuations from both the simulated trajectories and a distribution of crystallographic conformations. In both cases the main collective fluctuations describe domain motions. The protein consists of an N- and C-terminal domain connected by a long helix. The analysis of the distribution of crystallographic conformations reveals that the N-terminal helix rotates together with either of these two domains. The main domain fluctuation describes a closure mode of the two domains in which the N-terminal helix rotates concertedly with the C-terminal domain, while the domain fluctuation with second largest amplitude corresponds to a twisting mode of the two domains, with the N-terminal helix rotating concertedly with the N-terminal domain. For the closure mode, the difference in hinge-bending angle between the most open and most closed X-ray structure along this mode is 49 degrees. In the MD simulation that shows the largest fluctuation along this mode, a rotation of 45 degrees was observed. Although the twisting mode has much less freedom than the closure mode in the distribution of crystallographic conformations, experimental results suggest that it might be functionally important. Interestingly, the twisting mode is sampled more extensively in all MD simulations than it is in the distribution of X-ray conformations.

A comparison of a series of extended molecular dynamics (MD) simula- tions of bacteriophage T4 lysozyme in solvent with X-ray data is presented. Essential dynam- ics analyses were used to derive collective fluctuations from both the simulated trajecto- ries and a distribution of crystallographic con- formations. In both cases the main collective fluctuations describe domain motions. The pro- tein consists of an N- and C-terminal domain connected by a long helix. The analysis of the distribution of crystallographic conformations reveals that the N-terminal helix rotates to- gether with either of these two domains. The main domain fluctuation describes a closure mode of the two domains in which the N-termi- nal helix rotates concertedly with the C-termi- nal domain, while the domain fluctuation with second largest amplitude corresponds to a twisting mode of the two domains, with the N-terminal helix rotating concertedly with the N-terminal domain. For the closure mode, the difference in hinge-bending angle between the most open and most closed X-ray structure along this mode is 49 degrees. In the MD simu- lation that shows the largest fluctuation along this mode, a rotation of 45 degrees was ob- served. Although the twisting mode has much less freedom than the closure mode in the distribution of crystallographic conformations, experimental results suggest that it might be functionally important. Interestingly, the twist- ing mode is sampled more extensively in all MD simulations than it is in the distribution of X-ray conformations. Proteins 31:116-127

A method is presented that generates random protein structures that fulfil a set of upper and lower interatomic distance limits. These limits depend on distances measured in experimental structures and the strength of the interatomic interaction. Structural differences between generated structures are similar to those obtained from experiment and from MD simulation. Although detailed aspects of dynamical mechanisms are not covered and the extent of variations are only estimated in a relative sense, applications to an IgG-binding domain, an SH3 binding domain, HPr, calmodulin, and lysozyme are presented which illustrate the use of the method as a fast and simple way to predict structural variability in proteins. The method may be used to support the design of mutants, when structural fluctuations for a large number of mutants are to be screened. The results suggest that motional freedom in proteins is ruled largely by a set of simple geometric constraints.

A method is presented that generates random protein structures that fulfil a set of upper and lower interatomic distance limits. These limits depend on distances mea- sured in experimental structures and the strength of the interatomic interaction. Struc- tural differences between generated struc- tures are similar to those obtained from experi- ment and from MD simulation. Although detailed aspects of dynamical mechanisms are not covered and the extent of variations are only estimated in a relative sense, applications to an IgG-binding domain, an SH3 binding domain, HPr, calmodulin, and lysozyme are presented which illustrate the use of the method as a fast and simple way to predict structural variability in proteins. The method may be used to support the design of mutants, when structural fluctuations for a large num- ber of mutants are to be screened. The results suggest that motional freedom in proteins is ruled largely by a set of simple geometric constraints. Proteins 29:240-251, 1997. r 1997 Wiley-Liss, Inc.

Recently the basic theory of essential dynamics, a method for extracting large concerted motions from protein molecular dynamics trajectories, was described. Here, we introduce and test new aspects. A method for diagonalizing large covariance matrices is presented. We show that it is possible to perform essential dynamics using different subsets of atoms and compare these to the basic C-or analysis. Essential dynamics analyses are also compared to the normal modes method. The stability of the essential space during a simulation is investigated by comparing the two halves of a trajectory. Apart from the analyses in Cartesian space, the essential dynamics in phi/psi torsion angle space is discussed. (C) 1997 by John Wiley & Sons, Inc.

Recently, we developed a method (Amadei et al., J. Biomol. Str. Dyn. 13: 615-626; de Groot et al., J. Biomol. Str. Dyn. 13: 741-751, 1996) to obtain an extended sampling of the configurational space of proteins, using an adapted form of molecular dynamics (MD) simulations, based on the essential dynamics (ED) (Amadei et al., Proteins 17:412-425, 1993) method. In the present study, this ED sampling technique is applied to the histidine-containing phosphocarrier protein HPr from Escherichia coli. We find a cluster of conformations that is an order of magnitude larger than that found for a usual MD simulation of comparable length. The structures in this cluster are geometrically and energetically comparable to NMR structures. Moreover, on average, this large cluster satisfies nearly all NMR-derived distance restraints.

An exhaustive sampling of the configurational space of all-trans retinol using a 0.1 µs molecular-dynamics simulation is presented. The essential dynamics technique is used to describe the conformational changes in retinol using only three degrees of freedom. The different conformational states of retinol are analysed, and differences in free energy are calculated. The essential dynamics description allows a detailed comparison of free retinol and retinoids bound to retinoid-binding proteins and opens new possibilities in the small-molecule docking field. The dynamics of retinoids when complexed with their binding proteins are restricted, and they are forced into strained conformations. A ‘spring’ model for retinoid binding is proposed. This model is extended to a hypothesis for retinoid binding to visual pigments and bacteriorhodopsin.

The crystal structure of calcium-calmodulin (CaM) reveals a protein with a typical dumbbell structure. Various spectroscopic studies have suggested that the central linker region of CaM, which is alpha-helical in the crystal structure, is flexible in solution. In particular, NMR studies have indicated the presence of a flexible backbone between residues Lys 77 and Asp 80. This flexibility is related directly to the function of the protein because it enables the N- and C-terminal domains of the protein to move toward each other and bind to the CaM-binding domain of a target protein. We have investigated the flexibility of the CaM central helix by a variety of computational techniques: molecular dynamics (MD) simulations, normal mode analysis (NMA), and essential dynamics (ED) analysis. Our MD results reproduce the experimentally determined location of the bend in a simulation of only the CaM central helix, indicating that the bending point is an intrinsic property of the alpha-helix, for which the remainder of the protein is not important. Interestingly, the modes found by the ED analysis of the MD trajectory are very similar to the lowest frequency modes from the NM analysis and to modes found by an ED analysis of different structures in a set of NMR structures. Electrostatic interactions involving residues Arg 74 and Asp 80 seem to be important for these bending motions and unfolding, which is in line with pH-dependent NMR and CD studies.

The structure of the phosphorylated form of the histidine-containing phosphocarrier protein HPr from Escherichia coli has been solved by NMR and compared with that of unphosphorylated HPr. The structural changes that occur upon phosphorylation of His 15, monitored by changes in NOE patterns, 3JNHH alpha-coupling constants, and chemical shifts, are limited to the region around the phosphorylation site. The His15 backbone torsion angles become strained upon phosphorylation. The release of this strain during the phosphoryl-transfer to Enzyme II facilitates the transport of carbohydrates across the membrane. From an X-ray study of Streptococcus faecalis HPr (Jia Z, Vandonselaar M, Quail JW, Delbaere LTJ, 1993, Nature 361:94-97), it was proposed that the observed torsion-angle strain at residue 16 in unphosphorylated S. faecalis HPr has a role to play in the protein's phosphocarrier function. The model predicts that this strain is released upon phosphorylation. Our observations on E. coli HPr in solution, which shows strain only after phosphorylation, and the fact that all other HPrs studied thus far in their unphosphorylated forms show no strain either, led us to investigate the possibility that the crystal environment causes the strain in S. faecalis HPr. A 1-ns molecular dynamics simulation of S. faecalis HPr, under conditions that mimic the crystal environment, confirms the observations from the X-ray study, including the torsion-angle strain at residue 16. The strain disappeared, however, when S. faecalis HPr was simulated in a water environment, resulting in an active site configuration virtually the same as that observed in all other unphosphorylated HPrs. This indicates that the torsion-angle strain at Ala 16 in S. faecalis HPr is a result of crystal contacts or conditions and does not play a role in the phosphorylation-dephosphorylation cycle.

A method is presented for a more efficient sampling of the configurational space of proteins as compared to conventional sampling techniques such as molecular dynamics. The method is based on the large conformational changes in proteins revealed by the “essential dynamics” analysis. A form of constrained dynamics is performed, forcing the system to move along some of the essential coordinates. This results in a broader sampling of the essential subspace than in a comparable conventional molecular dynamics simulation without constraints. The new sampling method (essential dynamics sampling) was applied to the histidine-containing phosphocarrier protein HPr. The results indicate that the essential dynamics sampling method produces physically allowed structures, as estimated by the evaluation of many geometrical properties. In addition, a study of the motions in the essential subspace reveals a diffusion-like behavior.

We address the question how well proteins can be modelled on the basis of NMR data, when these data are incorporated into the protein model using distance restraints in a molecular dynamics simulation. We found, using HPr as a model protein, that distance restraining freezes the essential motion of proteins, as defined by Amadei et al. [Amadei, A., Linssen, A.B.M. and Berendsen, H.J.C. (1993) Protein Struct. Funct. Genet., 17, 412-425]. We discuss how modelling protocols can be improved in order to solve this problem.