Correlation of co-ordinated amino acid changes at the two-domain interface of cysteine proteases with protein stability.

The engineering of a protein containing an alternative local residue packing for a set of side-chains has proven to be a major challenge because compositional, volumetric and steric constraints must be respected. Homologous proteins should provide examples of alternative groups of residues leading to a similar functional result. The functional significance of a pair of co-ordinated changes that are observed in the cysteine proteases family has been investigated by comparing the effect of individual or double changes on secretion, stability and activity of papain. The two changes are not independent. Detrimental effects of single mutations at one of the two positions can be partly suppressed by the co-ordinated mutation that reproduces naturally occurring contacts, indicating that these changes are concerted. Single mutations at the other position produce milder effects, suggesting a pathway for evolution.

[1]  R. Sauer,et al.  An essential proline in lambda repressor is required for resistance to intracellular proteolysis. , 1990, Biochemistry.

[2]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[3]  I. G. Kamphuis,et al.  Thiol proteases. Comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain. , 1985, Journal of molecular biology.

[4]  P. Y. Chou,et al.  Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. , 1974, Biochemistry.

[5]  B. Cunningham,et al.  Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure. , 1987, Protein engineering.

[6]  W. Lim,et al.  Alternative packing arrangements in the hydrophobic core of λrepresser , 1989, Nature.

[7]  J. Knowles.  Tinkering with enzymes: what are we learning? , 1987, Science.

[8]  T. Kunkel.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J. A. Wozniak,et al.  Correlation between mutational destabilization of phage T4 lysozyme and increased unfolding rates. , 1991, Biochemistry.

[10]  I. G. Kamphuis,et al.  Structure of papain refined at 1.65 A resolution. , 1984, Journal of molecular biology.

[11]  L Serrano,et al.  Aromatic-aromatic interactions and protein stability. Investigation by double-mutant cycles. , 1991, Journal of molecular biology.

[12]  R. Sauer,et al.  Amino acid substitutions that increase the thermal stability of the λ Cro protein , 1989 .

[13]  C. H. Moore,et al.  The amino acid sequence of the tryptic peptides from actinidin, a proteolytic enzyme from the fruit of Actinidia chinensis. , 1978, The Biochemical journal.

[14]  K. Nagai,et al.  Coordinated amino acid changes in homologous protein families. , 1988, Protein engineering.

[15]  R. Sauer,et al.  Mutations in lambda repressor's amino-terminal domain: implications for protein stability and DNA binding. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Tadayuki Imanaka,et al.  A new way of enhancing the thermostability of proteases , 1986, Nature.

[17]  E N Baker,et al.  Structure of actinidin, after refinement at 1.7 A resolution. , 1980, Journal of molecular biology.

[18]  K. Nagai,et al.  Evolution of haemoglobin studied by protein engineering , 1988, BioEssays : news and reviews in molecular, cellular and developmental biology.

[19]  T C Terwilliger,et al.  Influence of interior packing and hydrophobicity on the stability of a protein. , 1989, Science.

[20]  Brian W. Matthews,et al.  Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing , 1990, Nature.

[21]  A. Lesk,et al.  Correlation of co-ordinated amino acid substitutions with function in viruses related to tobacco mosaic virus. , 1987, Journal of molecular biology.

[22]  D. Tessier,et al.  Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide. , 1991, Gene.

[23]  R. Ménard,et al.  A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain. , 1990, Biochemistry.

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

[25]  D. Tessier,et al.  Processing of the papain precursor. Purification of the zymogen and characterization of its mechanism of processing. , 1991, The Journal of biological chemistry.

[26]  M. Smith,et al.  Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. , 1982, Nucleic acids research.

[27]  J A Wozniak,et al.  Replacements of Pro86 in phage T4 lysozyme extend an alpha-helix but do not alter protein stability. , 1990, Science.

[28]  A. Fersht,et al.  COSMIC analysis of the major α-helix of barnase during folding , 1991 .

[29]  J. Wells,et al.  Additivity of mutational effects in proteins. , 1990, Biochemistry.

[30]  P Argos,et al.  Engineering protein thermal stability. Sequence statistics point to residue substitutions in alpha-helices. , 1989, Journal of molecular biology.

[31]  R. Pain,et al.  Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus. , 1991, Journal of molecular biology.

[32]  R. Jaenicke,et al.  Proteins under extreme physical conditions , 1990, FEBS letters.

[33]  B. Matthews,et al.  Hydrophobic packing in T4 lysozyme probed by cavity-filling mutants. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Tessier,et al.  Secretion of functional papain precursor from insect cells. Requirement for N-glycosylation of the pro-region. , 1990, The Journal of biological chemistry.

[35]  Alan R. Fersht,et al.  The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus) , 1984, Cell.

[36]  T. Alber,et al.  Mutational effects on protein stability. , 1989, Annual review of biochemistry.

[37]  D. Tessier,et al.  The expression in Escherichia coli of a synthetic gene coding for the precursor of papain is prevented by its own putative signal sequence. , 1989, Gene.

[38]  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.

[39]  A. Horovitz,et al.  Non-additivity in protein-protein interactions. , 1987, Journal of molecular biology.