Analysis of the effect of local interactions on protein stability.

BACKGROUND Protein stability appears to be governed by non-covalent interactions. These can be local (between residues close in sequence) or non-local (medium-range and long-range interactions). The specific role of local interactions is controversial. Statistical mechanics arguments point out that local interactions must be weak in stable folded proteins. However, site-directed mutagenesis has revealed that local interactions make a significant contribution to protein stability. Finally, computer simulations suggest that correctly folded proteins require a delicate balance between local and non-local contributions to protein stability. RESULT To analyze experimentally the effect of local interactions on protein stability, each of the five Che Y alpha-helices was enhanced in its helical propensity. alpha-Helix-promoting mutations have been designed, using a helix/coil transition algorithm tuned for heteropolypeptides, that do not alter the overall hydrophobicity or protein packing. The increase in helical propensity has been evaluated by far-UV CD analysis of the corresponding peptides. Thermodynamic analysis of the five Che Y mutants reveals, in all cases, an increase in half urea ([urea]1/2) and in Tm, and a decrease in the sensitivity to chemical denaturants (m). ANS binding assays indicate that the changes in m are not due to the stabilization of an intermediate, and the kinetic analysis of the mutants shows that their equilibrium unfolding transition can be considered as following a two-state model, while the change in m is found in the refolding reaction (m(k)f). CONCLUSIONS These results are explained by a variable two-state model in which the changes in half urea and Tm arise from the stabilization of the native state and the decrease in m from the compaction of the denatured state. Therefore, the net change in protein stability in aqueous solution produced by increasing the contribution of native-like local interactions in Che Y is the balance between these two conflicting effects. Our results support the idea that optimization of protein stability and cooperativity involve a specific ratio of local versus non-local interactions.

[1]  Jeremy M. Berg,et al.  Thermodynamic β -sheet propensities measured using a zinc-finger host peptide , 1993, Nature.

[2]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[3]  K. Dill,et al.  Cooperativity in protein-folding kinetics. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[6]  B. Lee,et al.  The interpretation of protein structures: estimation of static accessibility. , 1971, Journal of molecular biology.

[7]  L. Serrano,et al.  Thermodynamic analysis of the chemotactic protein from Escherichia coli, CheY. , 1993, Biochemistry.

[8]  V. Muñoz,et al.  Structural analysis of peptides encompassing all α-helices of three α/β parallel proteins: Che-Y, flavodoxin and P21-Ras: Implications for α-Helix stability and the folding of α/β parallel proteins , 1995 .

[9]  Luis Serrano,et al.  Elucidating the folding problem of helical peptides using empirical parameters , 1994, Nature Structural Biology.

[10]  R. L. Baldwin,et al.  The design and production of semisynthetic ribonucleases with increased thermostability by incorporation of S‐peptide analogues with enhanced helical stability , 1986, Proteins.

[11]  V. Muñoz,et al.  Kinetic characterization of the chemotactic protein from Escherichia coli, CheY. Kinetic analysis of the inverse hydrophobic effect. , 1994, Biochemistry.

[12]  K A Dill,et al.  Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation , 1993, Protein science : a publication of the Protein Society.

[13]  B. Matthews,et al.  Alanine scanning mutagenesis of the alpha-helix 115-123 of phage T4 lysozyme: effects on structure, stability and the binding of solvent. , 1995, Journal of molecular biology.

[14]  D. Shortle.  Staphylococcal nuclease: a showcase of m-value effects. , 1995, Advances in protein chemistry.

[15]  P. S. Kim,et al.  Measurement of the β-sheet-forming propensities of amino acids , 1994, Nature.

[16]  P. Matsumura,et al.  Crystal structure of Escherichia coli CheY refined at 1.7-A resolution. , 1993, The Journal of biological chemistry.

[17]  V. Muñoz,et al.  Elucidating the folding problem of helical peptides using empirical parameters. II. Helix macrodipole effects and rational modification of the helical content of natural peptides. , 1995, Journal of molecular biology.

[18]  D. Yee,et al.  Principles of protein folding — A perspective from simple exact models , 1995, Protein science : a publication of the Protein Society.

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

[20]  N. Kallenbach,et al.  Stabilization of myoglobin by multiple alanine substitutions in helical positions , 1994, Protein Science.

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

[22]  N. A. Rodionova,et al.  Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe , 1991, Biopolymers.

[23]  L Serrano,et al.  Helix design, prediction and stability. , 1995, Current opinion in biotechnology.

[24]  T. Kiefhaber,et al.  Kinetic coupling between protein folding and prolyl isomerization. I. Theoretical models. , 1992, Journal of molecular biology.

[25]  R. Jaenicke,et al.  Protein folding: local structures, domains, subunits, and assemblies. , 1991, Biochemistry.

[26]  A. Fersht,et al.  Alpha-helix stability in proteins. II. Factors that influence stability at an internal position. , 1992, Journal of molecular biology.

[27]  R. L. Baldwin,et al.  Intrinsic Stability of Individual α Helices Modulates Structure and Stability of the Apomyoglobulin Molten Globule Form , 1995 .

[28]  A. Fersht,et al.  Principles of protein stability derived from protein engineering experiments , 1993 .

[29]  L Regan,et al.  A thermodynamic scale for the beta-sheet forming tendencies of the amino acids. , 1994, Biochemistry.

[30]  R. Sauer,et al.  Mutational analysis of protein stability , 1992, Current Biology.

[31]  A. Fersht,et al.  Protein stability as a function of denaturant concentration: the thermal stability of barnase in the presence of urea. , 1995, Biochemistry.

[32]  J Skolnick,et al.  Computer modeling and folding of four‐helix bundles , 1993, Proteins.

[33]  K. Dill,et al.  Denatured states of proteins. , 1991, Annual review of biochemistry.

[34]  P. Lyu,et al.  Position-dependent stabilizing effects in .alpha.-helices: N-terminal capping in synthetic model peptides , 1992 .

[35]  Y H Chen,et al.  Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. , 1974, Biochemistry.