Isotopic labeling with hydrogen-2 and carbon-13 to compare conformations of proteins and mutants generated by site-directed mutagenesis, I

David W. Hibler, Lynn Harpold, Mark Dell'Acqua, Tayebeh Pourmotabbed, John A. Gerlt, Joyce A. Wilde, Philip H. Bolton

Research output: Contribution to journalArticlepeer-review

Abstract

This chapter describes the methods used for isotopic labeling of staphylococcal nuclease (SNase), as well as for one-dimensional nuclear magnetic resonance (NMR) characterization of the isotopically labeled samples. This chapter discusses the 1H and 13C NMR spectroscopy to compare the secondary and tertiary structures of wild-type and mutant variants created by site-directed mutagenesis. This chapter discusses one-dimensional 1H and 13C NMR spectra of labeled proteins. The effectiveness of the isotopic labeling procedure is determined by comparing the 1H and 13C NMR spectra of wild-type SNase and the site directed mutant E43D in which glutamate-43, the putative general base involved in the hydrolysis reaction, is replaced with aspartate. This mutant has a Vmax approximately 200-fold less and a Km approximately 8-fold greater than the values measured for the wild-type enzyme. The spectra of the isotopically labeled proteins convincingly demonstrate the power of deuteration of aromatic and aliphatic amino acid residues to accomplish residue-type assignments of resonances in the less congested regions of the 1H NMR spectrum and to ascertain the types of residues that undergo chemical shift changes when the site-directed substitution is accomplished.

Original languageEnglish (US)
Pages (from-to)74-86
Number of pages13
JournalMethods in enzymology
Volume177
Issue numberC
DOIs
StatePublished - Jan 1 1989
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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