Electrostatic stabilization of a thermophilic cold shock protein.

The cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus differs from its mesophilic homolog Bs-CspB from Bacillus subtilis by 15.8 kJ mol(-1) in the Gibbs free energy of denaturation (DeltaG(D)). The two proteins vary in sequence at 12 positions but only two of them, Arg3 and Leu66 of Bc-Csp, which replace Glu3 and Glu66 of Bs-CspB, are responsible for the additional stability of Bc-Csp. These two positions are near the ends of the protein chain, but close to each other in the three-dimensional structure. The Glu3Arg exchange alone changed the stability by more than 11 kJ mol(-1). Here, we elucidated the molecular origins of the stability difference between the two proteins by a mutational analysis. Electrostatic contributions to stability were characterized by measuring the thermodynamic stabilities of many variants as a function of salt concentration. Double and triple mutant analyses indicate that the stabilization by the Glu3Arg exchange originates from three sources. Improved hydrophobic interactions of the aliphatic moiety of Arg3 contribute about 4 kJ mol(-1). Another 4 kJ mol(-1) is gained from the relief of a pairwise electrostatic repulsion between Glu3 and Glu66, as in the mesophilic protein, and 3 kJ mol(-1) originate from a general electrostatic stabilization by the positive charge of Arg3, which is not caused by a pairwise interaction. Mutations of all potential partners for an ion pair within a radius of 10 A around Arg3 had only marginal effects on stability. The Glu3-->Arg3 charge reversal thus optimizes ionic interactions at the protein surface by both local and global effects. However, it cannot convert the coulombic repulsion with another Glu residue into a corresponding attraction. Avoidance of unfavorable coulombic repulsions is probably a much simpler route to thermostability than the creation of stabilizing surface ion pairs, which can form only at the expense of conformational entropy.

[1]  A. Horovitz,et al.  Double-mutant cycles: a powerful tool for analyzing protein structure and function. , 1996, Folding & design.

[2]  P. V. von Hippel,et al.  On the conformational stability of globular proteins. The effects of various electrolytes and nonelectrolytes on the thermal ribonuclease transition. , 1965, The Journal of biological chemistry.

[3]  U. Heinemann,et al.  Crystal structures of mutant forms of the Bacillus caldolyticus cold shock protein differing in thermal stability. , 2001, Journal of molecular biology.

[4]  P. Privalov,et al.  Energetics of protein structure. , 1995, Advances in protein chemistry.

[5]  B Honig,et al.  Free energy balance in protein folding. , 1995, Advances in protein chemistry.

[6]  R. Nussinov,et al.  Electrostatic strengths of salt bridges in thermophilic and mesophilic glutamate dehydrogenase monomers , 2000, Proteins.

[7]  M. Marahiel,et al.  Overproduction, crystallization, and preliminary X‐ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB , 1992, Proteins.

[8]  G. Makhatadze,et al.  Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability. , 1999, Biochemistry.

[9]  Hermann Schindelin,et al.  Universal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein , 1993, Nature.

[10]  Contributions of the ionizable amino acids to the stability of staphylococcal nuclease. , 1996 .

[11]  G. Böhm,et al.  The stability of proteins in extreme environments. , 1998, Current opinion in structural biology.

[12]  J. Reeve,et al.  Mutational Analysis of Differences in Thermostability between Histones from Mesophilic and Hyperthermophilic Archaea , 2000, Journal of bacteriology.

[13]  P. Harbury,et al.  Tanford-Kirkwood electrostatics for protein modeling. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[14]  B Honig,et al.  Electrostatic contributions to the stability of hyperthermophilic proteins. , 1999, Journal of molecular biology.

[15]  S L Mayo,et al.  Contribution of surface salt bridges to protein stability. , 2000, Biochemistry.

[16]  Interhelical ion pairing in coiled coils: solution structure of a heterodimeric leucine zipper and determination of pKa values of Glu side chains. , 2000, Biochemistry.

[17]  R. Sterner,et al.  Thermophilic Adaptation of Proteins , 2001, Critical reviews in biochemistry and molecular biology.

[18]  M Karplus,et al.  Enthalpic contribution to protein stability: insights from atom-based calculations and statistical mechanics. , 1995, Advances in protein chemistry.

[19]  A. Karshikoff,et al.  Optimization of the electrostatic interactions in proteins of different functional and folding type , 1994, Protein science : a publication of the Protein Society.

[20]  B. Tidor,et al.  Rational modification of protein stability by the mutation of charged surface residues. , 2000, Biochemistry.

[21]  J A McCammon,et al.  Molecular dynamics simulations of the hyperthermophilic protein sac7d from Sulfolobus acidocaldarius: contribution of salt bridges to thermostability. , 1999, Journal of molecular biology.

[22]  C. Pace Single surface stabilizer , 2000, Nature Structural Biology.

[23]  A. Fersht,et al.  Co-operative interactions during protein folding. , 1992, Journal of molecular biology.

[24]  R. L. Baldwin,et al.  How Hofmeister ion interactions affect protein stability. , 1996, Biophysical journal.

[25]  M. Record,et al.  Analysis of effects of salts and uncharged solutes on protein and nucleic acid equilibria and processes: a practical guide to recognizing and interpreting polyelectrolyte effects, Hofmeister effects, and osmotic effects of salts. , 1998, Advances in protein chemistry.

[26]  C. Tanford,et al.  Theory of Protein Titration Curves. I. General Equations for Impenetrable Spheres , 1957 .

[27]  J. Lebbink,et al.  Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface. , 1999, Journal of molecular biology.

[28]  Udo Heinemann,et al.  Two exposed amino acid residues confer thermostability on a cold shock protein , 2000, Nature Structural Biology.

[29]  A. Elcock The stability of salt bridges at high temperatures: implications for hyperthermophilic proteins. , 1998, Journal of molecular biology.

[30]  F. Arnold,et al.  Temperature adaptation of enzymes: lessons from laboratory evolution. , 2000, Advances in protein chemistry.

[31]  Kevin L. Shaw,et al.  Charge–charge interactions influence the denatured state ensemble and contribute to protein stability , 2000, Protein science : a publication of the Protein Society.

[32]  F. Schmid,et al.  In-vitro selection of highly stabilized protein variants with optimized surface. , 2001, Journal of molecular biology.

[33]  Kevin L. Shaw,et al.  Increasing protein stability by altering long‐range coulombic interactions , 1999, Protein science : a publication of the Protein Society.

[34]  B. Matthews Mutational analysis of protein stability , 1991 .

[35]  R. Jaenicke,et al.  Does the elimination of ion pairs affect the thermal stability of cold shock protein from the hyperthermophilic bacterium Thermotoga maritima? , 1999, FEBS letters.

[36]  G. Makhatadze,et al.  Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface. , 1999, Biochemistry.

[37]  Mohamed A. Marahiel,et al.  Conservation of rapid two-state folding in mesophilic, thermophilic and hyperthermophilic cold shock proteins , 1998, Nature Structural Biology.

[38]  P. Kraulis A program to produce both detailed and schematic plots of protein structures , 1991 .

[39]  M. Akke,et al.  Protein stability and electrostatic interactions between solvent exposed charged side chains , 1990, Proteins.

[40]  M. Perutz,et al.  Stereochemical basis of heat stability in bacterial ferredoxins and in haemoglobin A2 , 1975, Nature.

[41]  A. Karshikoff,et al.  Proteins from thermophilic and mesophilic organisms essentially do not differ in packing. , 1998, Protein engineering.

[42]  A. Fersht,et al.  Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles. , 1990, Biochemistry.

[43]  U. Hahn,et al.  Stability and folding kinetics of ribonuclease T1 are strongly altered by the replacement of cis-proline 39 with alanine. , 1993, Journal of molecular biology.

[44]  A. Horovitz,et al.  On the choice of reference mutant states in the application of the double-mutant cycle method. , 1996, Protein engineering.

[45]  J. Lebbink,et al.  Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. I. Introduction of a six-residue ion-pair network in the hinge region. , 1998, Journal of molecular biology.

[46]  T. Simonson,et al.  Macromolecular electrostatics: continuum models and their growing pains. , 2001, Current opinion in structural biology.

[47]  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.

[48]  A. Fersht,et al.  Surface electrostatic interactions contribute little of stability of barnase. , 1991, Journal of molecular biology.

[49]  D. Tobias,et al.  Electrostatics calculations: recent methodological advances and applications to membranes. , 2001, Current opinion in structural biology.

[50]  Y. Yamagata,et al.  Contribution of salt bridges near the surface of a protein to the conformational stability. , 2000, Biochemistry.

[51]  U Mueller,et al.  Thermal stability and atomic-resolution crystal structure of the Bacillus caldolyticus cold shock protein. , 2000, Journal of molecular biology.

[52]  M. Marahiel,et al.  Extremely rapid protein folding in the absence of intermediates , 1995, Nature Structural Biology.

[53]  M. Karplus,et al.  Electrostatic contributions to molecular free energies in solution. , 1998, Advances in protein chemistry.

[54]  M. Hennig,et al.  2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability. , 1995, Structure.

[55]  R. Jaenicke,et al.  The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima at 2.5 A resolution. , 1995, Journal of molecular biology.