Contribution of a proline residue and a salt bridge to the stability of a type I reverse turn in chymotrypsin inhibitor-2.
The contributions of the components of a type I reverse turn to the stability of chymotrypsin inhibitor-2 (Lys43-Pro44-Gly45) have been determined by protein engineering methods. A double-mutant cycle was used to determine the interaction between Lys43 and Glu45 by replacing them with alanine. We also mutated Pro44, which gives the geometry of the turn, to alanine and analysed the stability of the resulting mutants compared with wild-type chymotrypsin inhibitor-2, using equilibrium denaturation induced by guanidinium chloride. There are decreases in stability (in kcal/mol) of 0.64 +/- 0.06 for Lys43-->Ala, 0.57 +/- 0.15 for Glu45-->Ala, 0.95 +/- 0.06 for Lys43-->Ala/Glu45-->Ala and 1.93 +/- 0.09 for Pro44-->Ala. The free energy of interaction between Lys43 and Glu45 is calculated to be only 0.25 +/- 0.09 kcal/mol. From the changes in denaturation midpoint, Tm measured by circular dichroism, we estimate the energy of interaction between Lys43 and Glu45 to be 0.36 +/- 0.07 kcal/mol whereas the contribution of Pro44 is approximately 2.0 kcal/mol. The contribution of the salt bridge to the stability of the protein is very small and the residue Pro44 plays the key role in stabilizing the turn.
de Prat Gay
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MRC Unit for Protein Function and Design, Cambridge University Chemical Laboratory, UK.
Johnson
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CM
Fersht
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D006860:Hydrogen Bonding; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides; D010940:Plant Proteins / Q000737:chemistry*; D011392:Proline / Q000737:chemistry*; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary
D005971:Glutamates; D010455:Peptides; D010940:Plant Proteins; C093336:chymotrypsin inhibitor 2; D018698:Glutamic Acid; D011392:Proline; D008239:Lysine