Scientific Report 2006

Molecular Biology

Computer Modeling of Proteins and Nucleic Acids

D.A. Case, M. Crowley, Q. Cui, F. Dupradeau,* S. Moon, D. Nguyen, V. Pelmentschikov, D. Shivakumar, R.C. Walker, W. Zhang, J. Ziegler**

* Université Jules Verne, Amiens, France
** Universität Bayreuth, Bayreuth, Germany

Computer simulations offer an exciting approach to the study of many aspects of biochemical interactions. We focus primarily on molecular dynamics simulations (in which Newton’s equations of motions are solved numerically) to model the solution behavior of biomacromolecules. Recent applications include detailed analyses of electrostatic interactions in short peptides (folded and unfolded), proteins, and oligonucleotides in solution.

In addition, molecular dynamics methods are useful in refining solution structures of proteins by using constraints derived from nuclear magnetic resonance (NMR) spectroscopy, and we continue to explore new methods in this area. Our developments are incorporated into the Amber molecular modeling package, designed for large-scale biomolecular simulations, and into other software, including Nucleic Acid Builder, for developing 3-dimensional models of unusual nucleic acid structures; SHIFTS, for analyzing chemical shifts in proteins and nucleic acids; RNAmotif, for finding structural motifs in genomic sequence databases; and DOCK, for placing inhibitors into enzyme active sites.

NMR and the Structure and Dynamics of Proteins and Nucleic Acids

Our overall goal is to extract the maximum amount of information about biomolecular structure and dynamics from NMR experiments. To this end, we are studying the use of direct refinement methods for determining biomolecular structures in solution, going beyond distance constraints to generate closer connections between calculated and observed spectra. We are also using quantum chemistry to study chemical shifts and spin-spin coupling constants. Other types of data, such as chemical shift anisotropies, direct dipolar couplings in partially oriented samples, and analysis of cross-correlated relaxation, are also being used to guide structure refinement. In recent structural studies, we focused on the binding of zinc finger proteins with RNA and on structural influences on amide proton chemical shifts.

Nucleic Acid Modeling

Another project centers on the development of novel computer methods to construct models of “unusual” nucleic acids that go beyond traditional helical motifs. We are using these methods to study circular DNA, small RNA fragments, and 3- and 4-stranded DNA complexes, including models for recombination sites. We continue to develop efficient computer implementations of continuum solvent methods to allow simplified simulations that do not require a detailed description of the solvent (water) molecules; this approach also provides a useful way to study salt effects.

Recent efforts have made second derivatives of these energies available, so that normal mode analyses of nucleic acids with dozens to hundreds of nucleotides can be analyzed and the predictions compared with those of simpler, elastic continuum models. These efforts provide a new avenue for developing and testing low-resolution models that can be used for large molecular assemblies.

Dynamics and Energetics of Native and Nonnative States of Proteins

Analysis methods similar to those described for nucleic acids are also being used to estimate thermodynamic properties of “molten globules” and unfolded states of proteins. These studies are an extension of our earlier work on the folding of peptide fragments of proteins. A key feature is the development of computational methods that can be used to model pH and salt dependence of complex conformational transitions such as unfolding events. A second aspect of this research is a detailed interpretation of NMR results for protein nonnative states through molecular dynamics simulations and the construction of models for molecular motion and disorder.

All of these modeling activities are based on molecular mechanics force fields, which provide estimates of energies as a function of conformation. We continue to work on improvements in force fields; recently, we focused on adding aspects of electronic polarizability, going beyond the usual fixed-charge models, and on methods for handling arbitrary organic molecules that might be considered potential inhibitors in drug discovery efforts. Overall, the new models should provide a better picture of the noncovalent interactions between peptide groups and the groups’ surroundings, leading ultimately to more faithful simulations.

Vibrational Analysis of Iron-Sulfur Clusters in Proteins

A wide variety of proteins contain iron-sulfur clusters at their active sites; these proteins participate in electron-transport chains and in important enzymatic reactions such as the reduction of atmospheric nitrogen to ammonia by nitrogenase. Advances in synchrotron radiation sources now make it possible to probe the vibrational behavior of these clusters by using nuclear resonance vibrational spectroscopy (NRVS). This technique senses the coupling of a nuclear (Mossbauer) excitation to molecular vibrations. The result is a set of vibrational frequencies and intensities that indicate what sorts of deformations can take place. When the molecular structure is known, this information can contribute to the understanding of oxidation-reduction behavior and electron transfer kinetics. In situations in which the cluster structure is not known, NRVS data might useful as a “fingerprint” to help identify the structure.

We have been using quantum chemistry calculations to help understand NRVS spectra. Figure 1 shows a early example, comparing calculated and experimental spectra for a simple iron-sulfur “cubane” structure, a cluster type found in hundreds of known proteins. The calculations (shown as a dashed line) are in excellent agreement with experimental data (solid line), both in terms of frequencies and in terms of intensities. We are extending these calculations to models for the active site of nitrogenase, where the structure of the complex is still uncertain. If calculations like these can be used to closely track the experimental results, NRVS will be an important new tool for characterizing the active sites of metalloenzymes.

Fig. 1. Calculated and experimental NRVS spectra for an iron-sulfur cluster.

Publications

Baker, N.A., Bashford, D., Case, D.A. Implicit solvent electrostatics in biomolecular simulation. In: New Algorithms for Macromolecular Simulation. Leimkuhler, B., et al. (Eds.). Springer, New York, 2006, p. 263. Lecture Notes in Computational Science and Engineering, Vol. 49.

Brown, R.A., Case, D.A. Second derivatives in generalized Born theory. J. Comput. Chem. 27:1662, 2006.

Case, D.A., Cheatham, T.E. III, Darden, T., Gohlke, H., Luo, R., Merz, K.M., Jr., Onufriev, A., Simmerling, C., Wang, B., Woods, R. The Amber biomolecular simulation programs. J. Comput. Chem. 26:1668, 2005.

Dixit, S.B., Beveridge, D.L., Case, D.A., Cheatham, T.E. III, Giudice, E., Lankas, R., Lavery, R., Maddocks, J.H., Osman, R., Sklenar, H., Thayer, K.M., Varnai, P. Molecular dynamics simulations of the 136 unique tetranucleotide sequences of DNA oligonucleotides, II: sequence context effects on the dynamical structures of the 10 unique dinucleotide steps. Biophys. J. 89:3721, 2005.

Dupradeau, F.-Y., Case, D.A., Yu, C., Jimenez, R., Romesberg, F.E. Differential solvation and tautomer stability of a model base pair within the minor and major grooves of DNA. J. Am. Chem. Soc. 127:15612, 2005.

Lee, B.M., Xu, J., Clarkson, B.K., Martinez-Yamout, M.A., Dyson, H.J., Case, D.A., Gottesfeld, J.M., Wright, P.E. Induced fit and “lock and key” recognition of 5S RNA by zinc fingers of transcription factor IIIA. J. Mol. Biol. 357:275, 2006.

Mathews, D.H., Case, D.A. Nudged elastic band calculation of minimal energy pathways for the conformational change of a GG noncanonical pair. J. Mol. Biol. 357:1683, 2006.

Moon, S., Case, D.A. A comparison of quantum chemical models for calculating NMR shielding parameters in peptides: mixed basis set and ONIOM methods combined with a complete basis set extrapolation. J. Comput. Chem. 27:825, 2006.

Rizzo, R.C., Aynechi, T., Case, D.A., Kuntz, I.D. Estimation of absolute free energies of hydration using continuum methods: accuracy of partial charge models and optimization of nonpolar contributions. J. Chem. Theory Comput. 2:128, 2006.

Steinbrecher, T., Case, D.A., Labahn, A. A multistep approach to structure-based drug design: studying ligand binding at the human neutrophil elastase. J. Med. Chem. 49:1837, 2006.

Wang, J., Wang, W., Kollman, P.A., Case, D.A. Automatic atom type and bond type perception in molecular mechanical calculations. J. Mol. Graphics Model. 25:247, 2006.

Xiao, Y., Fisher, K., Smith, M.C., Newton, W.E., Case, D.A., George, S.J., Wang, H., Sturhahn, W., Alp, E.E., Zhao, J., Yoda, Y., Cramer, S.P. How nitrogenase shakes: initial information about P-cluster and FeMo-cofactor normal modes from nuclear resonance vibrational spectroscopy (NRVS). J. Am. Chem. Soc. 128:7608, 2006.

Xiao, Y., Koutmos, M., Case, D.A., Coucouvanis, D., Wang H., Cramer, S.P. Dynamics of an [Fe₄S₄(SPh)₄]^2– cluster via IR, Raman, and nuclear resonance vibrational spectroscopy (NRVS): analysis using ³⁶S substitution, DFT calculations, and empirical force fields. Dalton Trans. 2192, 2006, Issue 18.