Generation and analysis of proline mutants in protein G.

The pyrrolidine ring of the amino acid proline reduces the conformational freedom of the protein backbone in its unfolded form and thus enhances protein stability. The strategy of inserting proline into regions of the protein where it does not perturb the structure has been utilized to stabilize many different proteins including enzymes. However, most of these efforts have been based on trial and error, rather than rational design. Here, we try to understand proline's effect on protein stability by introducing proline mutations into various regions of the B1 domain of Streptococcal protein G. We also applied the Optimization of Rotamers By Iterative Techniques computational protein design program, using two different solvation models, to determine the extent to which it could predict the stabilizing and destabilizing effects of prolines. Use of a surface area dependent solvation model resulted in a modest correlation between the experimental free energy of folding and computed energies; on the other hand, use of a Gaussian solvent exclusion model led to significant positive correlation. Including a backbone conformational entropy term to the computational energies increases the statistical significance of the correlation between the experimental stabilities and both solvation models.

[1]  J. Thornton,et al.  Influence of proline residues on protein conformation. , 1991, Journal of molecular biology.

[2]  M. Karplus,et al.  Effective energy function for proteins in solution , 1999, Proteins.

[3]  W. Stites,et al.  Empirical evaluation of the influence of side chains on the conformational entropy of the polypeptide backbone , 1995, Proteins.

[4]  Ian W. Davis,et al.  Structure validation by Cα geometry: ϕ,ψ and Cβ deviation , 2003, Proteins.

[5]  B. Matthews,et al.  Flexible‐geometry conformational energy maps for the amino acid residue preceding a proline , 1992, Biopolymers.

[6]  R. H. Yun,et al.  Proline in α‐helix: Stability and conformation studied by dynamics simulation , 1991 .

[7]  S. L. Mayo,et al.  Conformational splitting: A more powerful criterion for dead‐end elimination , 2000, J. Comput. Chem..

[8]  A. Doig,et al.  Side-chain structures in the first turn of the alpha-helix. , 1999, Journal of molecular biology.

[9]  A. Gronenborn,et al.  A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. , 1993, Science.

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

[11]  P. S. Kim,et al.  Measurement of the β-sheet-forming propensities of amino acids , 1994, Nature.

[12]  F. Young Biochemistry , 1955, The Indian Medical Gazette.

[13]  Harold A. Scheraga,et al.  The Influence of Amino Acid Side Chains on the Free Energy of Helix-Coil Transitions1 , 1966 .

[14]  P J Flory,et al.  Conformational energies and configurational statistics of copolypeptides containing L-proline. , 1968, Journal of molecular biology.

[15]  Gunnar von Heijne,et al.  Proline kinks in transmembrane α-helices☆ , 1991 .

[16]  C. Vieille,et al.  Thermozymes: Identifying molecular determinants of protein structural and functional stability , 1996 .

[17]  S. L. Mayo,et al.  DREIDING: A generic force field for molecular simulations , 1990 .

[18]  A. Doig,et al.  Effect of the N1 residue on the stability of the α‐helix for all 20 amino acids , 2001, Protein science : a publication of the Protein Society.

[19]  K Watanabe,et al.  Multiple proline substitutions cumulatively thermostabilize Bacillus cereus ATCC7064 oligo-1,6-glucosidase. Irrefragable proof supporting the proline rule. , 1994, European journal of biochemistry.

[20]  S. L. Mayo,et al.  De novo protein design: fully automated sequence selection. , 1997, Science.

[21]  W. DeGrado,et al.  A thermodynamic scale for the helix-forming tendencies of the commonly occurring amino acids. , 1990, Science.

[22]  D. W. Bolen,et al.  Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. , 1988, Biochemistry.