Dynamic states of the three methionyl residues of Streptomyces subtilisin inhibitor. 1H NMR studies.
High resolution 1H NMR spectroscopy at 360 and 400 MHZ was used to study the dynamic states of the three methionyl residues (Met 70, 73, and 103) of Streptomyces subtilisin inhibitor (SSI) in a neutral aqueous (2H2O) solution. Chemical modification and nuclear Overhauser effect studies show that one of the methyl groups exists in the interior of the protein and is surrounded by phenylalanyl residues. With reference to the crystal structure, this methyl group is assignable to that of Met 103 in the hydrophobic core. The other two methionyl residues (Met 70 and 73) are equally susceptible to chemical modifications, but their methyl signals are affected in a different manner by the alpha-chymotrypsin-cleavage of the peptide bond between Met 73 and Val 74 at the reactive site. The observed linewidths of the methyl groups are consistent with the assumption that the whole side-chain of both Met 70 and 73 are already undergoing rapid internal motions at 20 degrees C, whereas for Met 103 only the rotation of the methyl group is allowed, even above approximately 40 degrees C. The high-field shift of the methyl proton resonance of Met 103 by about 0.3 ppm is attributable to the ring current effect from the surrounding phenylalanyl residues. This shift is invariant in the temperature range of 40-90 degrees C, showing that the native environment of the methyl group of Met 103 persists up to 90 degrees C at neutral pH. Destruction of the native methionyl environments occurs only above 90 degrees C, and the rate of conversion between the native and the denatured conformations is slower than 10 s-1. When SSI is mixed with subtilisin BPN' in a one-to-one molar ratio, at least one of the methyl signals of Met 70 and 73 is completely broadened, showing that the side-chain of this methionine is directly involved in the interaction with the enzyme.
D001426:Bacterial Proteins / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D002627:Chemistry, Physical; D002918:Chymotrypsin; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008715:Methionine / Q000032:analysis
D001426:Bacterial Proteins; C018150:subtilisin inhibitor protein, Streptomyces; D008715:Methionine; D002918:Chymotrypsin