Role of arginine 67 in the stabilization of chymotrypsin inhibitor 2: examination of amide proton exchange rates and denaturation thermodynamics of an engineered protein.
We have examined the contribution to protein stability of an interaction involving a charged hydrogen bond from an arginyl side chain (Arg67) in the serine proteinase inhibitor chymotrypsin inhibitor 2 (CI-2), by replacing this side chain with an alanyl residue by protein engineering. Using nuclear magnetic resonance spectroscopy (NMR), we have examined the effect of this mutation on the hydrogen-deuterium exchange rates of several backbone amide protons in the native and engineered proteins at 50 degrees C. These exchange rates provide a localized probe at multiple discrete sites throughout the protein and from comparison of native and mutant exchange rates allow calculation of the difference in free energy of exchange (delta delta Gex) resulting from the mutation. The results show that for the majority of amides observed this mutation results in delta delta Gex of ca. 1.7 kcal mol-1 over the whole CI-2 molecule. However, for two relatively exposed amide protons the exchange rates are found to be far less perturbed, implying that local unfolding mechanisms predominate for these protons. Direct measurement of the stability of both proteins to denaturation by guanidinum hydrochloride shows that the interaction contributes 1.4 kcal mol-1 to the stability of the molecule. This value is comparable to those obtained from the NMR exchange measurements and indicates that the exchange processes reflect the differences in stability between the native and mutant proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, U.K.
S K
SK
Jandu
S
S
Ray
L
L
Brooks
R J
RJ
Leatherbarrow
D001120:Arginine; D001483:Base Sequence; D001467:Hordeum; D006860:Hydrogen Bonding; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D015842:Serine Proteinase Inhibitors / Q000378:metabolism*; D013816:Thermodynamics
D010455:Peptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; C093336:chymotrypsin inhibitor 2; D001120:Arginine