Ruud M. Scheek

A protein structure from nuclear magnetic resonance data. lac repressor headpiece.

A procedure is described to determine the three-dimensional structure of biomolecules from nuclear magnetic resonance data. This procedure combines model building with a restrained molecular dynamics algorithm, in which distance information from nuclear Overhauser effects is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or headpiece (amino acid residues 1 to 51) of the lac repressor from Escherichia coli, for which no crystal structure is available. The relative orientation of the three helices of the headpiece is similar to that of the three homologous helices found in the cI repressor of bacteriophage lambda.

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.

Time-averaged nuclear overhauser effect distance restraints applied to tendamistat

A penalty function is introduced into molecular dynamics simulations that improves on current methods for enforcing nuclear magnetic resonance-based distance restraints. Rather than treating nuclear Overhauser effects as static distance bounds, they are considered as quantities that must be satisfied on average over the course of a simulation trajectory. The efficacy of the method is demonstrated on the previously determined structure of tendamistat. The molecular dynamics simulations show that the time-averaged constraints increase the mobility allowed to molecules, produce better agreement with distance bounds, improve searching properties and give a better estimate of the conformational space occupied by the molecule in solution.

TIME-DEPENDENT DISTANCE RESTRAINTS IN MOLECULAR-DYNAMICS SIMULATIONS

Abstract A method for enforcing nuclear Overhauser effect (NOE) distance restraints in molecular dynamics simulations is presented. Rather than model the NOE distance as static, a term is included in the force field such that the distance restraint need only be satisfied as a 〈 r −3 〉 −1/3 weighted time average over the simulation trajectory. This provides a better approximation of the physical nature of the NOE and reduces the disturbance to the force field due to the artificial term. Tests on a simple model system demonstrate the inadequacy of current methods and show the advantages of this novel approach, resulting in a more extensive search of conformational space.

Sequential assignment of imino- and amino-proton resonances in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy. Application to a lac operator fragment

A sequential procedure, based on 2D NOE spectroscopy, is described for identifying the resonances of water-exchangeable imino protons in NMR spectra of oligonucleotides. The adenine H2 and amino protons are also part of the cross-relaxation network of the imino protons and their resonance frequencies are easily found in the 2D NOE spectra. The NOE contacts between the amino and H5 protons of cytidines form an independent check for the correctness of the assignments, since these CHS protons are also part of the cross-relaxation network formed by the nonexchangeable protons, for which another sequential-assignment procedure exists (R. M. Scheek et al., J. Am. Chem. Soc.14, 2914 (1983)). The method was applied to a 14 bp lac operator fragment. The assignments of the resonances of the water-exchangeable imino and amino protons in this DNA fragment are required for studying its complex with the lac repressor headpiece.

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.

MOLECULAR-DYNAMICS SIMULATION TECHNIQUES FOR DETERMINATION OF MOLECULAR-STRUCTURES FROM NUCLEAR-MAGNETIC-RESONANCE DATA

Publisher Summary This chapter discusses the molecular dynamics (MD) simulation techniques for determination of molecular structures from nuclear magnetic resonance (NMR) data. This chapter presents with a short description of the MD technique and explain how the force field can be modified in order to bring or keep protons close together when a NOE between them has been measured. The combined use of distance geometry calculations and distance bounds driven dynamics gives insight into the accuracy of structure determinations by NMR. Refinement by restrained molecular dynamics improves the quality of the structures in terms of energetics. It is important in this respect to discriminate between uncertainties in the structure determination and thermal fluctuations. Computer simulation of at least the high-frequency modes of a molecules thermal motions by MD and calculation of the experimentally observed parameters by proper averaging over computed trajectories can be used to support a dynamic model, in which more than one “dynamic conformation” may have to be included to explain all experimental results.

The structure in solution of the b domain of protein disulfide isomerase

Protein disulfide isomerase (PDI) is a multifunctional protein of the endoplasmic reticulum, which catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. It consists of four domains designated a, b, b′ and a′. Both a and a′ domains contain an active site with the sequence motif -Cys-Gly-His-Cys- involved directly in thiol-disulfide exchange reactions. As expected these domains have structures very similar to the ubiquitous redox protein thioredoxin. A low-resolution NMR structure of the b domain revealed that this domain adopts a fold similar to the PDI a domain and thioredoxin [Kemmink, J., Darby, N.J., Dijkstra, K., Nilges, M. and Creighton, T.E. (1997) Curr. Biol., 7, 239–245]. A refined ensemble of solution structures based on the input of 1865 structural restraints shows that the structure of PDI b is well defined throughout the complete protein except for about 10 residues at the C-terminus of the sequence. 15N relaxation data show that these residues are disordered and not part of this structural domain. Therefore the domain boundaries of PDI can now be fixed with reasonable precision. Structural comparison of the PDI b domain with thioredoxin and PDI a reveals several features important for thiol-disulfide exchange activity.

The NMR determination of the IIAmtl binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system

The region of the surface of the histidine‐containing protein (HPr) which interacts with the A domain of the mannitol‐specific Enzyme II (IIAmtl) has been mapped by titrating the A‐domain into a solution of 15N‐labeled HPr and monitoring the effects on the amide proton and nitrogen chemical shifts via heteronuclear single quantum correlation spectroscopy (HSQC). Fourteen of the eighty‐five HPr amino acid residues show large changes in either the 15N or 1H chemical shifts or both as a result of the presence of IIAmtl while a further seventeen residues experience lesser shifts. Most of the residues involved are surface residues accounting for approximately 25% of the surface of HPr. Phosphorylation of HPr with catalytic amounts of Enzyme I (EI), in the absence of IIAmtl resulted in chemical shift changes in a sub‐set of the above residues; these were located more in the vicinity of the active site phospho‐histidine. Phosphorylation of the HPr/IIAmtl complex resulted in a HSQC spectrum which was indistinguishable from the P‐HPr spectrum in the absence of IIAmtl indicating that, as expected, the complex P‐HPr/P‐IIAmtl does not exist even at the high concentrations necessary for NMR.

Spatial arrangement of the three α helices in the solution conformation of E. coli lac represser DNA-binding domain

DNA‐binding protein 2D NMR Helix topology lac represser headpiece NOE‐distance constraint