Identification of residues involved in a conformational change accompanying substitutions for glutamate-43 in staphylococcal nuclease

Journal Article · Tue May 31 00:00:00 EDT 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00411a033· OSTI ID:6736274

Wilde, J A; Bolton, P H; Dell'Acqua, M; Hibler, D W; Pourmotabbed, T; Gerlt, J A

A recent paper from our laboratories 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, 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.

• Research Organization: Wesleyan Univ., Middletown, CT (USA)
• OSTI ID: 6736274
• Journal Information: Biochemistry; (United States), Vol. 27:11
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
NUCLEASES
MOLECULAR STRUCTURE
DEUTERIUM
GLUTAMIC ACID
NUCLEAR MAGNETIC RESONANCE
PLASMIDS
PROTONS
RESIDUES
STAPHYLOCOCCUS
TRACER TECHNIQUES
AMINO ACIDS
BACTERIA
BARYONS
CARBOXYLIC ACIDS
CELL CONSTITUENTS
ELEMENTARY PARTICLES
ENZYMES
ESTERASES
FERMIONS
HADRONS
HYDROGEN ISOTOPES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
RESONANCE
STABLE ISOTOPES
550201* - Biochemistry- Tracer Techniques