A computational approach for the rational design of stable proteins and enzymes: optimization of surface charge-charge interactions.

The design of stable proteins and enzymes is not only of particular biotechnological importance, but also addresses some important fundamental questions. While there are a number of different options available for designing or engineering stable proteins, the field of computational design provides fast and universal methods for stabilizing proteins of interest. One of the successful computational design strategies focuses on stabilizing proteins through the optimization of charge-charge interactions on the protein surface. By optimizing surface interactions, it is possible to alleviate some of the challenges that accompany efforts to redesign the protein core. The rational design of surface charge-charge interactions also allows one to optimize only the interactions that are distant from binding sites or active sites, making it possible to increase stability without adversely affecting activity. The optimization of surface charge-charge interactions is discussed in detail along with the experimental evidence to demonstrate that this is a robust and universal approach to designing proteins with enhanced stability.

[1]  D. Perl,et al.  Electrostatic stabilization of a thermophilic cold shock protein. , 2001, Journal of molecular biology.

[2]  B. Kuhlman,et al.  pKa values and the pH dependent stability of the N-terminal domain of L9 as probes of electrostatic interactions in the denatured state. Differentiation between local and nonlocal interactions. , 1999, Biochemistry.

[3]  R. Sauer,et al.  Contributions of a hydrogen bond/salt bridge network to the stability of secondary and tertiary structure in λ repressor , 1994, Protein science : a publication of the Protein Society.

[4]  C. Pace,et al.  Charge–charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa , 2005, Protein science : a publication of the Protein Society.

[5]  R. L. Baldwin,et al.  N‐ and C‐capping preferences for all 20 amino acids in α‐helical peptides , 1995, Protein science : a publication of the Protein Society.

[6]  Andrea Musacchio,et al.  High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides , 1994, Nature Structural Biology.

[7]  Srebrenka Robic,et al.  Energetic evidence for formation of a pH-dependent hydrophobic cluster in the denatured state of Thermus thermophilus ribonuclease H. , 2003, Journal of molecular biology.

[8]  A. Fersht,et al.  The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding. , 1992, Journal of molecular biology.

[9]  J R Desjarlais,et al.  De novo design of the hydrophobic cores of proteins , 1995, Protein science : a publication of the Protein Society.

[10]  J R Desjarlais,et al.  Side-chain and backbone flexibility in protein core design. , 1999, Journal of molecular biology.

[11]  B K Shoichet,et al.  A relationship between protein stability and protein function. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  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.

[13]  B. Matthews,et al.  Design and structural analysis of alternative hydrophobic core packing arrangements in bacteriophage T4 lysozyme. , 1993, Journal of molecular biology.

[14]  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.

[15]  Stephen L Mayo,et al.  Simple electrostatic model improves designed protein sequences , 2006, Protein science : a publication of the Protein Society.

[16]  L Serrano,et al.  The folding of an enzyme. II. Substructure of barnase and the contribution of different interactions to protein stability. , 1992, Journal of molecular biology.

[17]  A. Sali,et al.  Comparative protein structure modeling of genes and genomes. , 2000, Annual review of biophysics and biomolecular structure.

[18]  D. E. Anderson,et al.  pH-induced denaturation of proteins: a single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme. , 1990, Biochemistry.

[19]  M. Gilson,et al.  Prediction of pH-dependent properties of proteins. , 1994, Journal of molecular biology.

[20]  Hervé Minoux,et al.  An electrostatic basis for the stability of thermophilic proteins , 2004, Proteins.

[21]  Chie Motono,et al.  Thermodynamic and kinetic determinants of Thermotoga maritima cold shock protein stability: A structural and dynamic analysis , 2008, Proteins.

[22]  B. Stoddard,et al.  Computational Thermostabilization of an Enzyme , 2005, Science.

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

[24]  George I Makhatadze,et al.  Contribution of surface salt bridges to protein stability: guidelines for protein engineering. , 2003, Journal of molecular biology.

[25]  S. Marqusee,et al.  A thermodynamic comparison of mesophilic and thermophilic ribonucleases H. , 1999, Biochemistry.

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

[27]  G I Makhatadze,et al.  Major cold shock proteins, CspA from Escherichia coli and CspB from Bacillus subtilis, interact differently with single-stranded DNA templates. , 2000, Biochimica et biophysica acta.

[28]  F W Dahlquist,et al.  Thermodynamic basis for the increased thermostability of CheY from the hyperthermophile Thermotoga maritima. , 2001, Biochemistry.

[29]  Clare Woodward,et al.  Thermodynamics of bpti folding , 1993, Protein science : a publication of the Protein Society.

[30]  George I Makhatadze,et al.  Both helical propensity and side‐chain hydrophobicity at a partially exposed site in α‐helix contribute to the thermodynamic stability of ubiquitin , 2004, Proteins.

[31]  W. Kauzmann.  Some factors in the interpretation of protein denaturation. , 1959, Advances in protein chemistry.

[32]  J R Desjarlais,et al.  New strategies in protein design. , 1995, Current opinion in biotechnology.

[33]  James B. Matthew,et al.  [17] Calculation of electrostatic interactions in proteins , 1986 .

[34]  N R Kallenbach,et al.  Structural analysis of the N‐ and C‐termini in a peptide with consensus sequence , 1995, Protein science : a publication of the Protein Society.

[35]  B. Matthews,et al.  Core-packing constraints, hydrophobicity and protein design. , 1994, Current opinion in biotechnology.

[36]  F. Arnold,et al.  Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae. , 2001, Biotechnology and bioengineering.

[37]  M. Gilson,et al.  The determinants of pKas in proteins. , 1996, Biochemistry.

[38]  F. Avilés,et al.  The three‐dimensional structure of human procarboxypeptidase A2. Deciphering the basis of the inhibition, activation and intrinsic activity of the zymogen , 1997, The EMBO journal.

[39]  B K Shoichet,et al.  Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme. , 1999, Biochemistry.

[40]  George I Makhatadze,et al.  Role of the charge-charge interactions in defining stability and halophilicity of the CspB proteins. , 2007, Journal of molecular biology.

[41]  George I Makhatadze,et al.  Protein stability and surface electrostatics: a charged relationship. , 2006, Biochemistry.

[42]  Huan-Xiang Zhou,et al.  Similarity and difference in the unfolding of thermophilic and mesophilic cold shock proteins studied by molecular dynamics simulations. , 2006, Biophysical journal.

[43]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[44]  George I Makhatadze,et al.  Effects of charge-to-alanine substitutions on the stability of ribosomal protein L30e from Thermococcus celer. , 2005, Biochemistry.

[45]  Charles L Brooks,et al.  The effects of ionic strength on protein stability: the cold shock protein family. , 2002, Journal of molecular biology.

[46]  C. Pace,et al.  Forces contributing to the conformational stability of proteins , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[47]  L Serrano,et al.  Experimental analysis of the Schellman motif. , 1995, Journal of molecular biology.

[48]  George I Makhatadze,et al.  Removal of surface charge‐charge interactions from ubiquitin leaves the protein folded and very stable , 2002, Protein science : a publication of the Protein Society.

[49]  Nobutoshi Ito,et al.  Crystal structure at 1.92 Å resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin , 1994, Nature.

[50]  Kelly K. Lee,et al.  Distance dependence and salt sensitivity of pairwise, coulombic interactions in a protein , 2002, Protein science : a publication of the Protein Society.

[51]  P. Privalov,et al.  Thermodynamics of barnase unfolding , 1994, Protein science : a publication of the Protein Society.

[52]  J M Sturtevant,et al.  Cold denaturation of staphylococcal nuclease. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[53]  L Serrano,et al.  Thermodynamic analysis of helix-engineered forms of the activation domain of human procarboxypeptidase A2. , 2000, European journal of biochemistry.

[54]  J. Berg,et al.  Electrostatic interactions across a beta-sheet. , 1997, Biochemistry.

[55]  J A McCammon,et al.  Electrostatic contributions to the stability of halophilic proteins. , 1998, Journal of molecular biology.

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

[57]  Huan-Xiang Zhou,et al.  A Gaussian-chain model for treating residual charge–charge interactions in the unfolded state of proteins , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[58]  George I. Makhatadze,et al.  Thermodynamics Of α‐Helix Formation , 2005 .

[59]  B. García-Moreno E.,et al.  Salt effects on ionization equilibria of histidines in myoglobin. , 2000, Biophysical journal.

[60]  K. Dill.  Dominant forces in protein folding. , 1990, Biochemistry.

[61]  A. D. Robertson,et al.  Hydrogen bonds and the pH dependence of ovomucoid third domain stability. , 1995, Biochemistry.

[62]  Udo Heinemann,et al.  T-rich DNA single strands bind to a preformed site on the bacterial cold shock protein Bs-CspB. , 2006, Journal of molecular biology.

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

[64]  George I Makhatadze,et al.  Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge–charge interactions , 2007, Protein science : a publication of the Protein Society.

[65]  Arieh Warshel,et al.  Microscopic and semimicroscopic calculations of electrostatic energies in proteins by the POLARIS and ENZYMIX programs , 1993, J. Comput. Chem..

[66]  D C Rees,et al.  Some thermodynamic implications for the thermostability of proteins , 2001, Protein science : a publication of the Protein Society.

[67]  F Guarnieri,et al.  A self-consistent, microenvironment modulated screened coulomb potential approximation to calculate pH-dependent electrostatic effects in proteins. , 1999, Biophysical journal.

[68]  G. Makhatadze,et al.  Cold denaturation of ubiquitin. , 1999, Biochimica et biophysica acta.

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

[70]  D. Baker,et al.  A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins. , 2003, Journal of molecular biology.

[71]  George I. Makhatadze,et al.  Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C′ position of the C-capping box of α-helices , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[72]  Michael Wunderlich,et al.  Evolutionary protein stabilization in comparison with computational design. , 2005, Journal of molecular biology.

[73]  D. Baker,et al.  High-resolution Structural and Thermodynamic Analysis of Extreme Stabilization of Human Procarboxypeptidase by Computational Protein Design , 2007, Journal of molecular biology.

[74]  Rebecca Craig-Schapiro,et al.  Design of a hyperstable protein by rational consideration of unfolded state interactions. , 2006, Journal of the American Chemical Society.

[75]  Stephen L Mayo,et al.  Prudent modeling of core polar residues in computational protein design. , 2003, Journal of molecular biology.

[76]  G. Makhatadze,et al.  Interactions of the Major Cold Shock Protein of Bacillus subtilis CspB with Single-stranded DNA Templates of Different Base Composition* , 1999, The Journal of Biological Chemistry.

[77]  B. Honig,et al.  Classical electrostatics in biology and chemistry. , 1995, Science.

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

[79]  S. A. Marshall,et al.  Electrostatics significantly affect the stability of designed homeodomain variants. , 2002, Journal of molecular biology.

[80]  D. Raleigh,et al.  Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins. , 2005, Journal of molecular biology.

[81]  W A Hendrickson,et al.  Structure of a fibronectin type III domain from tenascin phased by MAD analysis of the selenomethionyl protein. , 1992, Science.

[82]  L Regan,et al.  An inverse correlation between loop length and stability in a four-helix-bundle protein. , 1997, Folding & design.

[83]  Stephen L Mayo,et al.  Evaluation of the energetic contribution of an ionic network to beta‐sheet stability , 2002, Protein science : a publication of the Protein Society.

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

[85]  P. Privalov,et al.  Thermodynamics of ubiquitin unfolding , 1994, Proteins.

[86]  P. Privalov,et al.  Cold Denaturation of Protein , 1990 .

[87]  G. Makhatadze,et al.  Heat capacity changes upon burial of polar and nonpolar groups in proteins , 2001, Protein science : a publication of the Protein Society.

[88]  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.

[89]  Duhee Bang,et al.  Dissecting the energetics of protein α-helix C-cap termination through chemical protein synthesis , 2006, Nature chemical biology.

[90]  G. A. Lazar,et al.  De novo design of the hydrophobic core of ubiquitin , 1997, Protein science : a publication of the Protein Society.

[91]  J A Wozniak,et al.  Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability. , 1991, Journal of molecular biology.

[92]  E. Alexov,et al.  Combining conformational flexibility and continuum electrostatics for calculating pK(a)s in proteins. , 2002, Biophysical journal.

[93]  Eugene I Shakhnovich,et al.  Physics and evolution of thermophilic adaptation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[94]  B K Shoichet,et al.  Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive. , 1995, Protein engineering.

[95]  G. Ramponi,et al.  Hydrolytic activity of muscle acyl phosphatase on 3-phosphoglyceryl phosphate , 1967, Experientia.

[96]  D. Laurents,et al.  pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1. , 1990, Biochemistry.

[97]  F. Gurd,et al.  Stabilization and destabilization of protein structure by charge interactions. , 1986, Methods in enzymology.

[98]  V L Arcus,et al.  pKA values of carboxyl groups in the native and denatured states of barnase: the pKA values of the denatured state are on average 0.4 units lower than those of model compounds. , 1995, Biochemistry.

[99]  A. Fersht,et al.  Strength and co-operativity of contributions of surface salt bridges to protein stability. , 1990, Journal of molecular biology.

[100]  F. Arnold,et al.  Directed evolution of a thermostable esterase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[101]  Michael Wunderlich,et al.  In Vitro Evolution of a Hyperstable Gβ1 Variant , 2006 .

[102]  A. Warshel,et al.  The effect of protein relaxation on charge-charge interactions and dielectric constants of proteins. , 1998, Biophysical journal.

[103]  C. Pace,et al.  Energetics of ribonuclease T1 structure. , 1994, Biochemistry.

[104]  Rajeev Aurora,et al.  Hydrophobic Interactions at the Ccap Position of the C-capping Motif of α-Helices , 2002 .

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

[106]  P. Nordlund,et al.  Crystal structure of common type acylphosphatase from bovine testis. , 1997, Structure.

[107]  Felix Franks,et al.  Conformational Stability of Proteins , 1993 .

[108]  B. García-Moreno E.,et al.  Structural interpretation of pH and salt-dependent processes in proteins with computational methods. , 2004, Methods in enzymology.

[109]  J. Parker.  Amino Acid Substitution , 2001 .

[110]  F. Gurd,et al.  pH-dependent processes in proteins. , 1985, CRC critical reviews in biochemistry.

[111]  George I Makhatadze,et al.  Thermodynamic consequences of burial of polar and non-polar amino acid residues in the protein interior. , 2002, Journal of molecular biology.

[112]  Michael Wunderlich,et al.  Stabilization of the cold shock protein CspB from Bacillus subtilis by evolutionary optimization of Coulombic interactions. , 2005, Journal of molecular biology.

[113]  E. Alexov,et al.  Incorporating protein conformational flexibility into the calculation of pH-dependent protein properties. , 1997, Biophysical journal.

[114]  C. Bugg,et al.  Structure of ubiquitin refined at 1.8 A resolution. , 1987, Journal of molecular biology.

[115]  A. Warshel,et al.  What are the dielectric “constants” of proteins and how to validate electrostatic models? , 2001, Proteins.

[116]  Jose M. Sanchez-Ruiz,et al.  Genetic Algorithm to Design Stabilizing Surface-Charge Distributions in Proteins , 2002 .

[117]  A. Warshel.  Computer simulations of enzyme catalysis: methods, progress, and insights. , 2003, Annual review of biophysics and biomolecular structure.

[118]  R. Jaenicke,et al.  Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima: catalytic, spectral and thermodynamic properties. , 1998, Journal of molecular biology.

[119]  A. Fersht,et al.  Importance of two buried salt bridges in the stability and folding pathway of barnase. , 1996, Biochemistry.

[120]  Axel T. Brünger,et al.  Amino-acid substitutions in a surface turn modulate protein stability , 1996, Nature Structural Biology.

[121]  K. Sharp,et al.  On the calculation of pKas in proteins , 1993, Proteins.

[122]  George I Makhatadze,et al.  Mechanism of thermostabilization in a designed cold shock protein with optimized surface electrostatic interactions. , 2004, Journal of molecular biology.

[123]  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.

[124]  Griko IuV,et al.  Cold denaturation of myoglobin in alkaline solutions , 1986 .

[125]  Christopher M. Summa,et al.  De novo design and structural characterization of proteins and metalloproteins. , 1999, Annual review of biochemistry.

[126]  Motowo Nakajima,et al.  Directed evolution by accumulating tailored mutations: thermostabilization of lactate oxidase with less trade-off with catalytic activity. , 2006, Protein engineering, design & selection : PEDS.

[127]  Huan-Xiang Zhou,et al.  Residual electrostatic effects in the unfolded state of the N-terminal domain of L9 can be attributed to nonspecific nonlocal charge-charge interactions. , 2002, Biochemistry.

[128]  Charles L. Brooks,et al.  How to improve nature: study of the electrostatic properties of the surface of α-lactalbumin , 2005 .

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

[130]  T M Handel,et al.  Review: protein design--where we were, where we are, where we're going. , 2001, Journal of structural biology.

[131]  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.

[132]  S. L. Mayo,et al.  Computational protein design. , 1999, Structure.

[133]  George I Makhatadze,et al.  Cooperativity of complex salt bridges , 2008, Protein science : a publication of the Protein Society.

[134]  A M Gronenborn,et al.  Thermodynamics of unfolding of the all beta-sheet protein interleukin-1 beta. , 1994, Biochemistry.

[135]  G. Makhatadze,et al.  Interactions of the cold shock protein CspB from Bacillus subtilis with single-stranded DNA. Importance of the T base content and position within the template. , 2001, The Journal of biological chemistry.

[136]  J M Sturtevant,et al.  Sidechain interactions in parallel beta sheets: the energetics of cross-strand pairings. , 1999, Structure.

[137]  P. Privalov,et al.  Heat and cold denaturation of phosphoglycerate kinase (interaction of domains) , 1989, FEBS letters.

[138]  D. Baker,et al.  Native protein sequences are close to optimal for their structures. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

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

[140]  Arieh Warshel,et al.  On the relationship between thermal stability and catalytic power of enzymes. , 2007, Biochemistry.

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

[142]  M. M. Garcia-Mira,et al.  Empirical parametrization of pK values for carboxylic acids in proteins using a genetic algorithm. , 2005, Biophysical chemistry.

[143]  R. L. Baldwin,et al.  Helix capping propensities in peptides parallel those in proteins. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[144]  I R Vetter,et al.  Nucleotide binding to the G12V‐mutant of Cdc42 investigated by X‐ray diffraction and fluorescence spectroscopy: Two different nucleotide states in one crystal , 2008, Protein science : a publication of the Protein Society.

[145]  B. Matthews,et al.  Studies on protein stability with T4 lysozyme. , 1995, Advances in protein chemistry.

[146]  E. Stellwagen,et al.  THERMOSTABILITY OF PROTEINS , 1978 .

[147]  Steven T. Whitten and,et al.  pH dependence of stability of staphylococcal nuclease: evidence of substantial electrostatic interactions in the denatured state. , 2000 .

[148]  L Serrano,et al.  Effect of active site residues in barnase on activity and stability. , 1992, Journal of molecular biology.

[149]  G. Makhatadze,et al.  To charge or not to charge? , 2001, Trends in biotechnology.

[150]  G. Makhatadze,et al.  Bacterial cold-shock proteins , 2002, Cellular and Molecular Life Sciences CMLS.

[151]  S. T. Thomas,et al.  Contribution of the 30/36 hydrophobic contact at the C-terminus of the alpha-helix to the stability of the ubiquitin molecule. , 2000, Biochemistry.

[152]  T. Richmond,et al.  Solvent accessible surface area and excluded volume in proteins. Analytical equations for overlapping spheres and implications for the hydrophobic effect. , 1984, Journal of molecular biology.

[153]  G. Ramponi,et al.  Aromatic acyl phosphates as substrates of acyl phosphatase. , 1966, Archives of biochemistry and biophysics.