Dissection of helix capping in T4 lysozyme by structural and thermodynamic analysis of six amino acid substitutions at Thr 59

Threonine 59, a helix-capping residue at the amino terminus of the longest helix in T4 phage lysozyme, was substituted with valine, alanine, glycine, serine, asparagine, and aspartic acid. The valine, alanine, and glycine replacements were observed to be somewhat more destabilizing than serine, asparagine, and aspartic acid. The crystal structures of the different variants showed that changes in conformation occurred at the site of substitution, including Asp 61, which is nearby, as well as displacement of a solvent molecule that is hydrogen-bonded to the gamma-oxygen of Thr 59 in wild-type lysozyme. Neither the structures nor the stabilities of the mutant proteins support the hypothesis of Serrano and Fersht (1989) that glycine and alanine are better helix-capping residues than valine because a smaller-sized residue allows better hydration at the end of the helix. In the aspartic acid and asparagine replacements the substituted side chains form hydrogen bonds with the end of the helix, as does threonine and serine at this position. In contrast, however, the Asp and Asn side chains also make unusually close contacts with carbon atoms in Asp 61. This suggests a structural basis for the heretofore puzzling observations that asparagine is more frequently observed as a helix-capping residue than threonine [Richardson, J. S., & Richardson, D. C. (1988) Science 240, 1648-1652] yet Thr----Asn replacements at N-cap positions in barnase were found to be destabilizing [Serrano, L., & Fersht, A. R. (1989) Nature 342, 296-299].(ABSTRACT TRUNCATED AT 250 WORDS)

[1]  E. Baker,et al.  Hydrogen bonding in globular proteins. , 1984, Progress in biophysics and molecular biology.

[2]  H. Berendsen,et al.  The α-helix dipole and the properties of proteins , 1978, Nature.

[3]  Tom Alber,et al.  Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozyme , 1988, Nature.

[4]  Robert L. Baldwin,et al.  Tests of the helix dipole model for stabilization of α-helices , 1987, Nature.

[5]  B. Matthews,et al.  Analysis of the interaction between charged side chains and the alpha-helix dipole using designed thermostable mutants of phage T4 lysozyme. , 1991, Biochemistry.

[6]  M. Goodman,et al.  Mechanisms of protein and polypeptide helix initiation , 1975 .

[7]  M. Smith,et al.  Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. , 1984, DNA.

[8]  B. Matthews,et al.  Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Alan R. Fersht,et al.  Capping and α-helix stability , 1989, Nature.

[10]  J. Richardson,et al.  Amino acid preferences for specific locations at the ends of alpha helices. , 1988, Science.

[11]  W. Baase,et al.  Thermal denaturation of bacteriophage T4 lysozyme at neutral pH. , 1987, Biopolymers.

[12]  Brian W. Matthews,et al.  Hydrophobic stabilization in T4 lysozyme determined directly by multiple substitutions of Ile 3 , 1988, Nature.

[13]  B. Matthews,et al.  A mutant T4 lysozyme (Val 131 → Ala) designed to increase thermostability by the reduction of strain within an α‐helix , 1990, Proteins.

[14]  D. E. Anderson,et al.  Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. , 1989, Methods in enzymology.

[15]  B. Matthews,et al.  Structure of a thermostable disulfide-bridge mutant of phage T4 lysozyme shows that an engineered cross-link in a flexible region does not increase the rigidity of the folded protein. , 1990, Biochemistry.

[16]  J. Ponder,et al.  Tertiary templates for proteins. Use of packing criteria in the enumeration of allowed sequences for different structural classes. , 1987, Journal of molecular biology.

[17]  J. Schellman,et al.  Thermodynamic stability and point mutations of bacteriophage T4 lysozyme. , 1984, Journal of molecular biology.

[18]  P. S. Kim,et al.  Nature of the charged-group effect on the stability of the C-peptide helix. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[19]  B. Matthews,et al.  Enhanced protein thermostability from designed mutations that interact with α-helix dipoles , 1990, Nature.

[20]  T. Ooi,et al.  Effects of salts on the nonequivalent stability of the α‐helices of isomeric block copolypeptides , 1982 .

[21]  Brian W. Matthews,et al.  An efficient general-purpose least-squares refinement program for macromolecular structures , 1987 .

[22]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[23]  T. A. Jones,et al.  A graphics model building and refinement system for macromolecules , 1978 .

[24]  L. J. Perry,et al.  Disulfide bonds and thermal stability in T4 lysozyme. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[25]  B. Matthews,et al.  Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths , 1991, Proteins.

[26]  B. Matthews,et al.  Structure of bacteriophage T4 lysozyme refined at 1.7 A resolution. , 1987, Journal of molecular biology.

[27]  W. J. Becktel,et al.  Protein stability curves , 1987, Biopolymers.

[28]  A. Bellocq,et al.  [Conformation analysis of L-pyroglutamic acid N-methylamide by depolarized Rayleigh diffusion, vibration spectroscopy, and conformational energy calculation]. , 1975, Biopolymers.

[29]  B. Matthews,et al.  Structure and thermal stability of phage T4 lysozyme. , 1987, Methods in enzymology.