The molecular basis for the chemical denaturation of proteins by urea

Molecular dynamics simulations of the protein chymotrypsin inhibitor 2 in 8 M urea at 60°C were undertaken to investigate the molecular basis of chemical denaturation. The protein unfolded rapidly under these conditions, but it retained its native structure in a control simulation in water at the same temperature. The overall process of unfolding in urea was similar to that observed in thermal denaturation simulations above the protein's Tm of 75°C. The first step in unfolding was expansion of the hydrophobic core. Then, the core was solvated by water and later by urea. The denatured structures in both urea and at high temperature contained residual native helical structure, whereas the β-structure was completely disrupted. The average residence time for urea around hydrophilic groups was six times greater than around hydrophobic residues and in all cases greater than the corresponding water residence times. Water self-diffusion was reduced 40% in 8 M urea. Urea altered water structure and dynamics, thereby diminishing the hydrophobic effect and encouraging solvation of hydrophobic groups. In addition, through urea's weakening of water structure, water became free to compete with intraprotein interactions. Urea also interacted directly with polar residues and the peptide backbone, thereby stabilizing nonnative conformations. These simulations suggest that urea denatures proteins via both direct and indirect mechanisms.

[1]  Ron Elber,et al.  The dominant interaction between peptide and urea is electrostatic in nature: A molecular dynamics simulation study , 2003, Biopolymers.

[2]  M. Record,et al.  Thermal and urea-induced unfolding of the marginally stable lac repressor DNA-binding domain: a model system for analysis of solute effects on protein processes. , 2003, Biochemistry.

[3]  M. Record,et al.  Generalized derivation of an exact relationship linking different coefficients that characterize thermodynamic effects of preferential interactions. , 2002, Biophysical chemistry.

[4]  S. N. Timasheff.  Thermodynamic binding and site occupancy in the light of the Schellman exchange concept. , 2002, Biophysical chemistry.

[5]  V. Daggett,et al.  Increasing temperature accelerates protein unfolding without changing the pathway of unfolding. , 2002, Journal of molecular biology.

[6]  S. N. Timasheff,et al.  Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Schellman,et al.  Fifty years of solvent denaturation. , 2002, Biophysical chemistry.

[8]  A. Fersht,et al.  Protein Folding and Unfolding at Atomic Resolution , 2002, Cell.

[9]  Valerie Daggett,et al.  The molecular mechanism of stabilization of proteins by TMAO and its ability to counteract the effects of urea. , 2002, Journal of the American Chemical Society.

[10]  M. Record,et al.  Thermodynamics of interactions of urea and guanidinium salts with protein surface: Relationship between solute effects on protein processes and changes in water‐accessible surface area , 2001, Protein science : a publication of the Protein Society.

[11]  Valerie Daggett,et al.  Protein folding from a highly disordered denatured state: The folding pathway of chymotrypsin inhibitor 2 at atomic resolution , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Saraste,et al.  FEBS Lett , 2000 .

[13]  A Caflisch,et al.  Structural details of urea binding to barnase: a molecular dynamics analysis. , 1999, Structure.

[14]  Kenneth M. Merz,et al.  Rationalization of the enantioselectivity of subtilisin in DMF , 1999 .

[15]  A. Fersht,et al.  Synergy between simulation and experiment in describing the energy landscape of protein folding. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[16]  K. P. Murphy,et al.  Urea effects on protein stability: Hydrogen bonding and the hydrophobic effect , 1998, Proteins.

[17]  S. N. Timasheff,et al.  Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated. , 1998, Advances in protein chemistry.

[18]  D. W. Bolen,et al.  A naturally occurring protective system in urea-rich cells: mechanism of osmolyte protection of proteins against urea denaturation. , 1997, Biochemistry.

[19]  Michael Levitt,et al.  Calibration and Testing of a Water Model for Simulation of the Molecular Dynamics of Proteins and Nucleic Acids in Solution , 1997 .

[20]  J Tirado-Rives,et al.  Molecular dynamics simulations of the unfolding of barnase in water and 8 M aqueous urea. , 1997, Biochemistry.

[21]  D. Hartsough,et al.  Solvation and Dynamics of Chymotrypsin in Hexane , 1996 .

[22]  V. Daggett,et al.  Testing the Modified Hydration-Shell Hydrogen-Bond Model of Hydrophobic Effects Using Molecular Dynamics Simulation , 1996 .

[23]  A. Li,et al.  Identification and characterization of the unfolding transition state of chymotrypsin inhibitor 2 by molecular dynamics simulations. , 1996, Journal of molecular biology.

[24]  A. Fersht,et al.  Structure of the transition state for folding of a protein derived from experiment and simulation. , 1996, Journal of molecular biology.

[25]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[26]  M. Levitt,et al.  Potential energy function and parameters for simulations of the molecular dynamics of proteins and nucleic acids in solution , 1995 .

[27]  K Wüthrich,et al.  Interaction of urea with an unfolded protein The DNA‐binding domain of the 434‐repressor , 1995, FEBS letters.

[28]  V. Daggett,et al.  Molecular dynamics simulations of protein unfolding and limited refolding: characterization of partially unfolded states of ubiquitin in 60% methanol and in water. , 1995, Journal of molecular biology.

[29]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[30]  A. Li,et al.  Characterization of the transition state of protein unfolding by use of molecular dynamics: chymotrypsin inhibitor 2. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[31]  A. Fersht,et al.  Direct observation of better hydration at the N terminus of an alpha-helix with glycine rather than alanine as the N-cap residue. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[32]  S. N. Timasheff,et al.  The control of protein stability and association by weak interactions with water: how do solvents affect these processes? , 1993, Annual review of biophysics and biomolecular structure.

[33]  S. N. Timasheff.  Water as ligand: preferential binding and exclusion of denaturants in protein unfolding. , 1992, Biochemistry.

[34]  P. Privalov,et al.  Protein interactions with urea and guanidinium chloride. A calorimetric study. , 1992, Journal of molecular biology.

[35]  A. Fersht,et al.  Folding of chymotrypsin inhibitor 2. 1. Evidence for a two-state transition. , 1991, Biochemistry.

[36]  K. Dill,et al.  Solvent denaturation and stabilization of globular proteins. , 1991, Biochemistry.

[37]  Thomas E. Creighton,et al.  Stability of folded conformations , 1991 .

[38]  Norbert Muller,et al.  Search for a realistic view of hydrophobic effects , 1990 .

[39]  J. Schellman.  Selective binding and solvent denaturation , 1987, Biopolymers.

[40]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[41]  T. Arakawa,et al.  Protein stabilization and destabilization by guanidinium salts. , 1984, Biochemistry.

[42]  T. Arakawa,et al.  Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding. , 1984, Biochemistry.

[43]  Kazimierz Krynicki,et al.  Pressure and temperature dependence of self-diffusion in water , 1978 .

[44]  R. C. Weast.  Handbook of chemistry and physics , 1973 .

[45]  P. V. Hippel,et al.  Ion effects on the solution structure of biological macromolecules , 1969 .

[46]  C. Tanford.  Protein denaturation. , 1968, Advances in protein chemistry.

[47]  G. Kell,et al.  Precise representation of volume properties of water at one atmosphere , 1967 .

[48]  W. Jencks,et al.  THE EFFECT OF COMPOUNDS OF THE UREA-GUANIDINIUM CLASS ON THE ACTIVITY COEFFICIENT OF ACETYLTETRAGLYCINE ETHYL ESTER AND RELATED COMPOUNDS. , 1965, Journal of the American Chemical Society.

[49]  C. Tanford,et al.  The unfolding of beta-lactoglobulin at pH 3 by urea, formamide, and other organic substances. , 1961, The Journal of biological chemistry.

[50]  W. Kauzmann.  Some factors in the interpretation of protein denaturation. , 1959, Advances in protein chemistry.

[51]  C. Tomes.  CHEMISTRY AND PHYSICS , 1903 .