Identification of residues involved in a conformational change accompanying substitutions for glutamate-43 in staphylococcal nuclease.
A recent paper from our laboratories [Hibler, D. W., Stolowich, N. J., Reynolds, M. A., Gerlt, J. A. Wilde, J. A., & Bolton, P. H. (1987) Biochemistry 26, 6278] described the generation of site-directed substitutions for the putative general base Glu-43 in the active site of Staphylococcal nuclease (SNase) and the use of 1H NMR spectroscopy to characterize the effect of the substitutions on the conformations of the mutant proteins. The replacements for Glu-43 (Asp, Gln, Asn, Ser, and Ala) both decreased the catalytic efficiency and changed the one- and two-dimensional NMR spectral properties of the mutant enzymes. We have prepared and studied the NMR spectral properties of several samples of deuteriated wild-type SNase that allow sequence-specific resonance assignments for several aromatic and aliphatic amino acid side chains that experience changes both in normal one-dimensional spectra and in two-dimensional NOESY spectra. Due to severe spectral congestion of resonances in the one- and two-dimensional spectra of protiated SNase, the assignments would have been difficult, if not impossible, to obtain without deuteriation of selected amino acids. The spectra we have obtained demonstrate that changes in NOE intensities involve a valine residue that is spatially adjacent to two phenylalanine residues; given the X-ray structure for SNase [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551], these residues must be Val-74, Phe-34, and Phe-76. In addition, a leucine residue experiencing changes in NOE intensities spatially adjacent to Val-74 and Phe-34 can be assigned to Leu-25.(ABSTRACT TRUNCATED AT 250 WORDS)
Wilde
J A
JA
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06457.
Bolton
P H
PH
Dell'Acqua
M
M
Hibler
D W
DW
Pourmotabbed
T
T
Gerlt
J A
JA
D001665:Binding Sites; D003903:Deuterium; D005971:Glutamates; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011522:Protons
D005971:Glutamates; D011522:Protons; D003903:Deuterium; D008836:Micrococcal Nuclease