Protein stabilization by hydrophobic interactions at the surface.

The contribution of the solvent-exposed residue 63 to thermal stability of the thermolysin-like neutral protease of Bacillus stearothermophilus was studied by analyzing the effect of twelve different amino acid substitutions at this position. The thermal stability of the enzyme was increased considerably by introducing Arg, Lys or bulky hydrophobic amino acids. In general, the effects of the mutations showed that hydrophobic contacts in this surface-located region of the protein are a major determinant of thermal stability. This observation contrasts with general concepts concerning the contribution of surface-located residues and surface hydrophobicity to protein stability and indicates new ways for protein stabilization by site-directed mutagenesis.

[1]  A. Fujishima,et al.  Electrochemical Photolysis of Water at a Semiconductor Electrode , 1972, Nature.

[2]  A. Fontana.  Structure and stability of thermophilic enzymes. Studies on thermolysin. , 1988, Biophysical chemistry.

[3]  M Bolognesi,et al.  Grafting of a calcium-binding loop of thermolysin to Bacillus subtilis neutral protease. , 1991, Biochemistry.

[4]  B. Matthews,et al.  The conformation of thermolysin. , 1974, The Journal of biological chemistry.

[5]  J A Wells,et al.  Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'. , 1992, Biochemistry.

[6]  T. Imanaka,et al.  Nucleotide sequence and promoter region for the neutral protease gene from Bacillus stearothermophilus , 1985, Journal of bacteriology.

[7]  G. Vriend,et al.  Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines , 1993, FEBS letters.

[8]  B. van den Burg,et al.  A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product , 1991, Journal of bacteriology.

[9]  G. Vriend,et al.  Structural Determinants of the Thermostability of thermolysin-Like Bacillus Neutral Proteases , 1993 .

[10]  R. Heinrikson.  Applications of thermolysin in protein structural analysis. , 1977, Methods in Enzymology.

[11]  R. Heinrikson.  [20] Applications of thermolysin in protein structural analysis , 1977 .

[12]  W. Lim,et al.  Deciphering the message in protein sequences: tolerance to amino acid substitutions. , 1990, Science.

[13]  F. Dahlquist,et al.  Role of Calcium in the thermal stability of thermolysin. , 1976, Biochemistry.

[14]  S. Bouvier,et al.  Systematic mutation of bacteriophage T4 lysozyme. , 1991, Journal of molecular biology.

[15]  A. Lesk,et al.  Determinants of a protein fold. Unique features of the globin amino acid sequences. , 1987, Journal of molecular biology.

[16]  K. Wilson,et al.  The structure of neutral protease from Bacillus cereus at 0.2-nm resolution. , 1992, European journal of biochemistry.

[17]  G. Vriend,et al.  STABILITY AND STABILIZATION OF ENZYMES , 1993 .

[18]  B. Matthews,et al.  Structural and genetic analysis of protein stability. , 1993, Annual review of biochemistry.

[19]  B. van den Burg,et al.  One-step affinity purification of Bacillus neutral proteases using bacitracin-silica. , 1989, Journal of biochemical and biophysical methods.

[20]  M. Bolognesi,et al.  Structural features of neutral protease from Bacillus subtilis deduced from model-building and limited proteolysis experiments. , 1990, European journal of biochemistry.

[21]  B. Matthews,et al.  Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein. , 1987, Biochemistry.

[22]  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.

[23]  B. van den Burg,et al.  Identification of autodigestion target sites in Bacillus subtilis neutral proteinase. , 1990, The Biochemical journal.

[24]  K. Titani,et al.  Amino-acid sequence of thermolysin. , 1972, Nature: New biology.

[25]  G Vriend,et al.  Effects of changing the interaction between subdomains on the thermostability of Bacillus neutral proteases , 1992, Proteins.

[26]  B. Matthews,et al.  Structural basis of amino acid alpha helix propensity. , 1993, Science.

[27]  M. L. Connolly.  Solvent-accessible surfaces of proteins and nucleic acids. , 1983, Science.

[28]  B P Schoenborn,et al.  Three-dimensional structure of thermolysin. , 1972, Nature: New biology.

[29]  G. Vriend,et al.  Thermostability of Bacillus subtilis neutral protease. , 1991, Biochemistry international.

[30]  J. Jenkins,et al.  Crystal structure of neutral protease from Bacillus cereus refined at 3.0 A resolution and comparison with the homologous but more thermostable enzyme thermolysin. , 1988, Journal of molecular biology.

[31]  M. Perutz,et al.  Molecular pathology of human haemoglobin. , 1968, Nature.

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

[33]  R. Sauer,et al.  Functionally acceptable substitutions in two alpha-helical regions of lambda repressor. , 1990, Proteins.

[34]  T. Creighton,et al.  Protein Folding , 1992 .

[35]  Gert Vriend,et al.  Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases , 1993, J. Comput. Aided Mol. Des..

[36]  B. Matthews,et al.  Structure of thermolysin refined at 1.6 A resolution. , 1982, Journal of molecular biology.

[37]  G Vriend,et al.  WHAT IF: a molecular modeling and drug design program. , 1990, Journal of molecular graphics.

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

[39]  C. Chothia,et al.  Hydrophobic bonding and accessible surface area in proteins , 1974, Nature.

[40]  T. Imanaka,et al.  Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis , 1983, Journal of bacteriology.

[41]  Robert T. Sauer,et al.  Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface , 1990, Nature.