B. L. de Groot

PREDICTION OF PROTEIN CONFORMATIONAL FREEDOM FROM DISTANCE 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 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. Proteins 29:240–251, 1997.

DOMAIN MOTIONS IN BACTERIOPHAGE T4 LYSOZYME : A COMPARISON BETWEEN MOLECULAR DYNAMICS AND CRYSTALLOGRAPHIC DATA

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. Proteins 31:116–127, 1998.

Mechanism of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase - Structures of complexes with the substrate

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniaehyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-Å resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn349, His399, and Tyr408, is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the α- and β-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme.

A comparison of techniques for calculating protein essential dynamics

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‐α 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 ϕ/ψ torsion angle space is discussed.

The consistency of large concerted motions in proteins in molecular dynamics simulations

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.

Normal Modes and Essential Dynamics

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.

An extended sampling of the configurational space of HPr from E. coli.

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.

Progress in the analysis of membrane protein structure and function

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 sub‐nanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress.

Towards an exhaustive sampling of the configurational spaces of the two forms of the peptide hormone guanylin

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.

Kinetics of Conformational Sampling in Ubiquitin

Molecular recognition plays a central role in many biological processes. For enzymatic reactions and slow protein–protein recognition events, turn-over rates and on-rates in the millisecond-to-second time scale have been connected to internal protein dynamics detected with atomic resolution by NMR spectroscopy, and in particular conformational sampling could be established as a mechanism for enzyme–substrate and protein–protein recognition. Recent theoretical studies indicate that faster rates of conformational interconversion in the microsecond time scale might limit on-rates for protein–protein recognition. However experimental proofs were lacking so far, mainly because such rates could not be determined accurately enough and kinetic experiments in the microsecond time range are difficult to perform. Nevertheless, for proteins and TAR-RNA, recent studies based on residual dipolar couplings (RDCs) and other NMR spectroscopy techniques have detected substantial internal dynamics in a time window from the rotational correlation time tc (one-digit nanoseconds) to approximately 50 ms, called the supra-tc window in the following. However, the exact rates of internal dynamics within this four orders of magnitude wide time window could not be determined. Supra-tc dynamics in ubiquitin [9] and TAR-RNA could be connected to the conformational sampling required for molecular recognition. While the amplitudes of motions have been indirectly detected by RDCs and characterized in great detail, it has so far been impossible to directly observe these motions and to determine the exact rate of these supra-tc motions. In contrast, conformational sampling in enzymes occurs on a time scale that is 100 to 1000 times slower than supra-tc dynamics and therefore NMR relaxation dispersion (RD) techniques have been able to establish the functional link to enzyme kinetics with atomic resolution at physiological conditions. 5] However, for technical reasons, RD is not sensitive to motion faster than approximately 50 ms (RD window) and therefore does not access motion in the supra-tc window at room temperature. Here we determine the rate of interconversion between conformers of free ubiquitin by a combination of NMR RD experiments in super-cooled solution and dielectric relaxation spectroscopy (DR). Furthermore, we corroborate the motional amplitudes in the RDC-derived ensembles quantitatively with the observed amplitudes of RD and DR. The methods utilized herein can be used to directly study protein dynamics in a time range that was previously inaccessible. Significant motional amplitude in the supra-tc window has been observed using RDC measurements, and was connected to the conformational sampling for a protein in the ground