Biomedical subjects

Charles D Schwieters

Publications and source records attributed to Charles D Schwieters.

10 recordsLinked to original sources

PubMed2006

Concordance of residual dipolar couplings, backbone order parameters and crystallographic B-factors for a small alpha/beta protein: a unified picture of high probability, fast atomic motions in proteins.

Using ensemble refinement of the third immunoglobulin binding domain (GB3) of streptococcal protein G (a small alpha/beta protein of 56 residues), we demonstrate that backbone (N-H, N-C', Calpha-Halpha, Calpha-C') residual dipolar coupling data in five independent alignment media, generalized order parameters from 15N relaxation data, and B-factors from a high-resolution (1.1A), room temperature crystal structure are entirely consistent with one another within experimental error. The optimal ensemble size representation is between four and eight, as assessed by complete cross-validation of the residual dipolar couplings. Thus, in the case of GB3, all three observables reflect the same low-amplitude anisotropic motions arising from fluctuations in backbone phi/psi torsion angles in the picosecond to nanosecond regime in both solution and crystalline environments, yielding a unified picture of fast, high-probability atomic motions in proteins. An understanding of these motions is crucial for understanding the impact of protein dynamics on protein function, since they provide part of the driving force for triggered conformational changes that occur, for example, upon ligand binding, signal transduction and enzyme catalysis.

Bacterial Proteins↗

PubMed2004

Characterization of nonspecific protein-DNA interactions by 1H paramagnetic relaxation enhancement.

Nonspecific protein-DNA interactions play an important role in a variety of contexts related to DNA packaging, nucleoprotein complex formation, and gene regulation. Biophysical characterization of nonspecific protein-DNA interactions at the atomic level poses significant challenges owing to the dynamic nature of such complexes. Although NMR spectroscopy represents a powerful tool for the analysis of dynamic systems, conventional NMR techniques have provided little information on nonspecific protein-DNA interactions. We show that intermolecular (1)H paramagnetic relaxation enhancement (PRE) arising from Mn(2+) chelated to an EDTA-group covalently attached to a thymine base (dT-EDTA-Mn(2+)) in DNA provides a unique approach for probing the global dynamics and equilibrium distribution of nonspecific protein-DNA interactions. For nonspecific DNA binding, similar intermolecular (1)H-PRE profiles are observed on the (1)H resonances of the bound protein when dT-EDTA-Mn(2+) is located at either end of a DNA oligonucleotide duplex. We demonstrate the applicability of this approach to HMG-box proteins and contrast the results obtained for nonspecific DNA binding of the A-box of HMGB-1 (HMGB-1A) with sequence-specific DNA binding of the related SRY protein. Intermolecular (1)H-PRE data demonstrate unambiguously that HMGB-1A binds to multiple sites in multiple orientations even on a DNA fragment as short as 14 base pairs. Combining the (1)H-PRE data with the crystal structure of the HMGB-1 A-box/cisplatin-modified DNA complex allows one to obtain a semiquantitative estimate of the equilibrium populations at the various sites.

Amino Acid Sequence↗

PubMed2004

Amplitudes of protein backbone dynamics and correlated motions in a small alpha/beta protein: correspondence of dipolar coupling and heteronuclear relaxation measurements.

Backbone residual dipolar coupling (N-H, Calpha-Halpha, N-C', and Calpha-C') data collected in five different media on the B3 IgG binding domain of streptococcal protein G (GB3) have been analyzed by simultaneous refinement of the coordinates and optimization of the magnitudes and orientations of the alignment tensors using single and multiple structure representations. We show, using appropriate error analysis, that agreement between observed and calculated dipolar couplings at the level of experimental uncertainty is obtained with a two-structure (N(e) = 2) ensemble representation which represents the simplest equilibrium description of anisotropic motions. The data permit one to determine the magnitude of the anisotropic motions along the four different backbone bond vectors in terms of order parameters. The order parameters, , for the N-H bond vectors are in qualitative agreement with the generalized order parameters, S(2)NH(relaxation), derived from (15)N relaxation measurements, with a correlation coefficient of 0.84. S(2)NH(relaxation) can be regarded as the product of an anisotropic order parameter, corresponding to derived from the residual dipolar couplings, and an axially symmetric order parameter, S(2)NH(axial), corresponding to bond librations which are expected to be essentially uniform along the polypeptide chain. The current data indicate that the average value of S(2)NH(axial) is approximately 0.9. The close correspondence of and S(2)NH(relaxation) indicates that any large-scale displacements from the mean coordinate positions on time scales longer than the rotational correlation time are rare and hence do not perturb the observed dipolar couplings. Analysis of a set of 100 N(e) = 2 ensembles reveals the presence of some long-range correlated motions of N-H and Calpha-Halpha vectors involving residues far apart in the sequence but close together in space. In addition, direct evidence is obtained for ubiquitous crankshaft motions along the entire length of the polypeptide backbone manifested by the anticorrelation of the backbone torsion angles phi(i) and psi(i-1).

Anisotropy↗

PubMed2004

Completely automated, highly error-tolerant macromolecular structure determination from multidimensional nuclear overhauser enhancement spectra and chemical shift assignments.

The major rate-limiting step in high-throughput NMR protein structure determination involves the calculation of a reliable initial fold, the elimination of incorrect nuclear Overhauser enhancement (NOE) assignments, and the resolution of NOE assignment ambiguities. We present a robust approach to automatically calculate structures with a backbone coordinate accuracy of 1.0-1.5 A from datasets in which as much as 80% of the long-range NOE information (i.e., between residues separated by more than five positions in the sequence) is incorrect. The current algorithm differs from previously published methods in that it has been expressly designed to ensure that the results from successive cycles are not biased by the global fold of structures generated in preceding cycles. Consequently, the method is highly error tolerant and is not easily funnelled down an incorrect path in either three-dimensional structure or NOE assignment space. The algorithm incorporates three main features: a linear energy function representation of the NOE restraints to allow maximization of the number of simultaneously satisfied restraints during the course of simulated annealing; a method for handling the presence of multiple possible assignments for each NOE cross-peak which avoids local minima by treating each possible assignment as if it were an independent restraint; and a probabilistic method to permit both inactivation and reactivation of all NOE restraints on the fly during the course of simulated annealing. NOE restraints are never removed permanently, thereby significantly reducing the likelihood of becoming trapped in a false minimum of NOE assignment space. The effectiveness of the algorithm is demonstrated using completely automatically peak-picked experimental NOE data from two proteins: interleukin-4 (136 residues) and cyanovirin-N (101 residues). The limits of the method are explored using simulated data on the 56-residue B1 domain of Streptococcal protein G.

Algorithms↗

PubMed2004

Ensemble approach for NMR structure refinement against (1)H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule.

Paramagnetic relaxation enhancement (PRE) measurements on (1)H nuclei have the potential to play an important role in NMR structure determination of macromolecules by providing unique long-range (10-35 A) distance information. Recent methodological advances for covalently attaching paramagnetic groups at specific sites on both proteins and nucleic acids have permitted the application of the PRE to various biological macromolecules. However, because artificially introduced paramagnetic groups are exposed to solvent and linked to the macromolecule by several freely rotatable bonds, they are intrinsically flexible. This renders conventional back-calculation of the (1)H-PRE using a single-point representation inaccurate, thereby severely limiting the utility of the (1)H-PRE as a tool for structure refinement. To circumvent these limitations, we have developed a theoretical framework and computational strategy with which to accurately back-calculate (1)H-PREs arising from flexible paramagnetic groups attached to macromolecules. In this scheme, the (1)H-PRE is calculated using a modified Solomon-Bloembergen equation incorporating a "model-free" formalism, based on a multiple-structure representation of the paramagnetic group in simulated annealing calculations. The ensemble approach for (1)H-PRE back-calculation was examined using several SRY/DNA complexes incorporating dT-EDTA-Mn(2+) at three distinct sites in the DNA, permitting a large data set comprising 435 experimental backbone and side-chain (1)H-PREs to be obtained in a straightforward manner from 2D through-bond correlation experiments. Calculations employing complete cross-validation demonstrate that the ensemble representation provides a means to accurately utilize backbone and side-chain (1)H-PRE data arising from a flexible paramagnetic group in structure refinement. The results of (1)H-PRE based refinement, in conjunction with previously obtained NMR restraints, indicate that significant gains in accuracy can be readily obtained. This is particularly significant in the case of macromolecular complexes where intermolecular translational restraints derived from nuclear Overhauser enhancement data may be limited.

Base Sequence↗

PubMed2004

How much backbone motion in ubiquitin is required to account for dipolar coupling data measured in multiple alignment media as assessed by independent cross-validation?

The magnitude of backbone internal motions in the small protein ubiquitin that needs to be invoked to account for dipolar coupling data measured in multiple alignment media is investigated using an intuitively straightforward approach. This involves simultaneous refinement of the coordinates (against NOE, torsion angle, and dipolar coupling restraints) and optimization of the magnitudes and orientations of the alignment tensors by means of torsion angle simulated annealing and Cartesian space minimization. We show that N-H dipolar couplings in 11 different alignment media and N-C', H(N)-C', and C alpha-C' dipolar coupling in two alignment media can be accounted for, at approximately the level of uncertainty in the experimental data, by a single structure representation. Extension to a two-member ensemble representation which provides the simplest description of anisotropic motions in the form of a two-site jump model (in which the overall calculated dipolar couplings are the averages of the calculated dipolar couplings of the individual ensemble members), results in modest, but significant, improvements in dipolar coupling R-factors for both the working set of couplings used in the refinement and for the free cross-validated set of C alpha-H alpha dipolar couplings recorded in two alignment media. Extensions to larger ensemble sizes do not result in any R-factor improvement for the cross-validated C alpha-H alpha dipolar couplings. With a few notable exceptions, the amplitudes of the anisotropic motions are small, with S(2)(jump) order parameters > or =0.8. Moreover, the structural impact of those few residues that do exhibit larger amplitude motions (S(2)(jump) ranging from 0.3 to 0.8) is minimal and can readily be accommodated by very small backbone atomic rms shifts (<0.5 A) because of compensatory changes in phi and psi backbone torsion angles. In addition, evidence for correlated motions of N-H bond vectors is observed. For most practical applications, however, refinement of NMR structures against dipolar couplings using a single structure representation is adequate and will not adversely impact coordinate accuracy within the limits of the NMR method.

Kinetics↗

PubMed2003

Docking of protein-protein complexes on the basis of highly ambiguous intermolecular distance restraints derived from 1H/15N chemical shift mapping and backbone 15N-1H residual dipolar couplings using conjoined rigid body/torsion angle dynamics.

A simple and reliable method for docking protein-protein complexes using (1)H(N)/(15)N chemical shift mapping and backbone (15)N-(1)H residual dipolar couplings is presented and illustrated with three complexes (EIN-HPr, IIA(Glc)-HPr, and IIA(Mtl)-HPr) of known structure. The (1)H(N)/(15)N chemical shift mapping data are transformed into a set of highly ambiguous, intermolecular distance restraints (comprising between 400 and 3000 individual distances) with translational and some degree of orientational information content, while the dipolar couplings provide information on relative protein-protein orientation. The optimization protocol employs conjoined rigid body/torsion angle dynamics in simulated annealing calculations. The target function also comprises three nonbonded interactions terms: a van der Waals repulsion term to prevent atomic overlap, a radius of gyration term (E(rgyr)) to avoid expansion at the protein-protein interface, and a torsion angle database potential of mean force to bias interfacial side chain conformations toward physically allowed rotamers. For the EIN-HPr and IIA(Glc)-HPr complexes, all structures satisfying the experimental restraints (i.e., both the ambiguous intermolecular distance restraints and the dipolar couplings) converge to a single cluster with mean backbone coordinate accuracies of 0.7-1.5 A. For the IIA(Mtl)-HPr complex, twofold degeneracy remains, and the structures cluster into two distinct solutions differing by a 180 degrees rotation about the z axis of the alignment tensor. The correct and incorrect solutions which have mean backbone coordinate accuracies of approximately 0.5 and approximately 10.5 A, respectively, can readily be distinguished using a variety of criteria: (a) examination of the overall (1)H(N)/(15)N chemical shift perturbation map (because the incorrect cluster predicts the presence of residues at the interface that experience only minimal chemical shift perturbations; this information is readily incorporated into the calculations in the form of ambiguous intermolecular repulsion restraints); (b) back-calculation of dipolar couplings on the basis of molecular shape; or (c) the E(rgyr) distribution which, because of its global nature, directly reflects the interfacial packing quality. This methodology should be particularly useful for high throughput, NMR-based, structural proteomics.

Bacterial Proteins↗

PubMed2003

The Xplor-NIH NMR molecular structure determination package.

We announce the availability of the Xplor-NIH software package for NMR biomolecular structure determination. This package consists of the pre-existing XPLOR program, along with many NMR-specific extensions developed at the NIH. In addition to many features which have been developed over the last 20 years, the Xplor-NIH package contains an interface with a new programmatic framework written in C++. This interface currently supports the general purpose scripting languages Python and TCL, enabling rapid development of new tools, such as new potential energy terms and new optimization methods. Support for these scripting languages also facilitates interaction with existing external programs for structure analysis, structure manipulation, visualization, and spectral analysis.

Molecular Structure↗

PubMed2002

Theoretical and computational advances in biomolecular NMR spectroscopy.

Recent developments in experimental and computational aspects of NMR spectroscopy have had a significant impact on the accuracy and speed of macromolecular structure determination in solution, particularly with regard to systems of high complexity (such as protein complexes). These include experiments designed to provide long-range orientational and translational restraints, improvements in internal coordinate dynamics used for simulated annealing, and the development of database potentials of mean force to improve the description of the non-bonded contacts.

Databases, Factual↗

PubMed2002

Reweighted atomic densities to represent ensembles of NMR structures.

A reweighted atomic probability density is introduced as a means of representing ensembles of NMR structures in a simple, concise and informative manner. This density is shown to give a better visual representation of molecular structure information than an unweighted density, and should provide a useful interactive graphics tool during the course of iterative NMR structure refinement. The approach is illustrated using several examples.

Animals↗