EFFECTS OF REPLACEMENT OF LYS25 WITH GLN ON THE CONFORMATION OF RIBONUCLEASE T-1 - SEQUENCE-SPECIFIC H-1-NMR RESONANCE ASSIGNMENTS OF GLN25 RIBONUCLEASE T-1 BY 2-DIMENSIONAL NMR-SPECTROSCOPY

Two isozymes of ribonuclease (RNase) T-1 exist in nature, i.e., Gln25 RNase T-1 and Lys25 RNase T-1. Gln25 RNase T-1 is less stable than Lys25 RNase T-1, although the enzymatic activity is not distinguishable between these two isozymes. To elucidate the effects of the replacement of Lys25 with Gin on the conformation and microenvironments of RNase T-1 in detail, two-dimensional NMR spectra were measured, sequence-specific H-1 NMR resonance assignments of Gln25 RNase T-1 were performed, and then the determined parameters and microenvironments of Gln25 RNase T-1 were compared with those of Lys25 isozyme [Hoffmann, E, and Ruterjans, H. (1988) fur. J. Biochem. 177, 539-580]. The main chain protons were assigned for 101 out of the total of 104 amino acid residues. Secondary structure elements were identified from analysis of characteristic NOE patterns, interstrand NOE connectivities, and hydrogen-deuterium exchange rates of main chain amide protons. The results indicated that Gln25 RNase T-1 contains a single alpha-helix and seven beta-strands. The secondary structure of Gln25 RNase T-1 is, thus, essentially the same as that of Lys25 RNase T-1. On the other hand, comparison of the conformation-dependent shifts of Gln25 RNase T-1 with those of Lys25 RNase T-1 showed that the replacement of Lys25 with Gin has significant effects on the C-terminal part of the alpha-helix region and the base-binding site. These results may indicate that the base-binding site is relatively flexible in the RNase T(?)1 molecule. Among the residues of the C-terminal part of the alpha-helix region, the protons of Asp29 were most affected in terms of their chemical shifts, which may indicate that the side chain carboxylate anion of Asp29 is the counterpart of the electrostatic interaction of Lys25 in Lys25 RNase T-1. The Gln25 of Gln25 RNase T-1 may have little or no interaction with Asp29, and this may be the reason why Gln25 RNase T-1 is less stable than the Lys25 isozyme.