Free-energy calculations highlight differences in accuracy between X-ray and NMR structures and add value to protein structure prediction.

BACKGROUND While X-ray crystallography structures of proteins are considerably more reliable than those from NMR spectroscopy, it has been difficult to assess the inherent accuracy of NMR structures, particularly the side chains. RESULTS For 15 small single-domain proteins, we used a molecular mechanics-/dynamics-based free-energy approach to investigate native, decoy, and fully extended alpha conformations. Decoys were all less energetically favorable than native conformations in nine of the ten X-ray structures and in none of the five NMR structures, but short 150 ps molecular dynamics simulations on the experimental structures caused them to have the lowest predicted free energy in all 15 proteins. In addition, a strong correlation exists (r(2) = 0.86) between the predicted free energy of unfolding, from native to fully extended conformations, and the number of residues. CONCLUSIONS This work suggests that the approximate treatment of solvent used in solving NMR structures can lead NMR model conformations to be less reliable than crystal structures. This conclusion was reached because of the considerably higher calculated free energies and the extent of structural deviation during aqueous dynamics simulations of NMR models compared to those determined by X-ray crystallography. Also, the strong correlation found between protein length and predicted free energy of unfolding in this work suggests, for the first time, that a free-energy function can allow for identification of the native state based on calculations on an extended state and in the absence of an experimental structure.

[1]  D. Baker,et al.  Molecular dynamics in the endgame of protein structure prediction. , 2001, Journal of molecular biology.

[2]  Charles L. Brooks,et al.  Identifying native‐like protein structures using physics‐based potentials , 2002, J. Comput. Chem..

[3]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[4]  F E Cohen,et al.  Protein model structure evaluation using the solvation free energy of folding. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[5]  K. Dill.  Theory for the folding and stability of globular proteins. , 1985, Biochemistry.

[6]  D. Agard,et al.  Two energetically disparate folding pathways of α-lytic protease share a single transition state , 2000, Nature Structural Biology.

[7]  P. Wolynes,et al.  Spin glasses and the statistical mechanics of protein folding. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[8]  P. Kollman,et al.  A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules , 1995 .

[9]  M R Lee,et al.  Use of MM‐PB/SA in estimating the free energies of proteins: Application to native, intermediates, and unfolded villin headpiece , 2000, Proteins.

[10]  M. Karplus,et al.  Discrimination of the native from misfolded protein models with an energy function including implicit solvation. , 1999, Journal of molecular biology.

[11]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .

[12]  A. Pohorille,et al.  The development/application of a ‘minimalist’ organic/biochemical molecular mechanic force field using a combination of ab initio calculations and experimental data , 1997 .

[13]  P. Kollman,et al.  Continuum Solvent Studies of the Stability of DNA, RNA, and Phosphoramidate−DNA Helices , 1998 .

[14]  Richard Bonneau,et al.  Ab initio protein structure prediction of CASP III targets using ROSETTA , 1999, Proteins.

[15]  P. Kollman,et al.  Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. , 2000, Accounts of chemical research.

[16]  M. Levitt,et al.  Energy functions that discriminate X-ray and near native folds from well-constructed decoys. , 1996, Journal of molecular biology.

[17]  D. Baker,et al.  2.1 and 1.8 Å Average Cα RMSD Structure Predictions on Two Small Proteins, HP-36 and S15 , 2001 .

[18]  S. Bryant,et al.  Statistics of sequence-structure threading. , 1995, Current opinion in structural biology.

[19]  M. Karplus,et al.  Effective energy functions for protein structure prediction. , 2000, Current opinion in structural biology.

[20]  P. Weiner,et al.  Computer Simulation of Biomolecular Systems , 1997 .

[21]  C. Anfinsen.  Principles that govern the folding of protein chains. , 1973, Science.

[22]  S Vajda,et al.  Discrimination of near‐native protein structures from misfolded models by empirical free energy functions , 2000, Proteins.

[23]  A. D. McLachlan,et al.  Solvation energy in protein folding and binding , 1986, Nature.

[24]  T. Darden,et al.  Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems , 1993 .

[25]  K. Dill,et al.  From Levinthal to pathways to funnels , 1997, Nature Structural Biology.

[26]  E S Huang,et al.  Factors affecting the ability of energy functions to discriminate correct from incorrect folds. , 1997, Journal of molecular biology.

[27]  Gillian Rhodes.  Crystallography Made Crystal Clear , 1993 .