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

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

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

This chapter discusses the use of isotopic labeling to assign the aromatic partners of cross-peaks of both wild-type and mutant proteins through the use of deuteration of both aliphatic and aromatic amino acid side chains. Comparison of the data for isotopically labeled wild-type and mutant proteins allow independent assignment of the cross-peaks in these proteins. This contribution will conclude with a description of how the side chain assignments can be extended to site-specific assignments. For a protein the size of Snase, 149 amino acids plus a hexapeptide tail in the form one study, methods that depend on resolved scalar couplings or on sufficient resolution of the two-dimensional data will typically not be entirely adequate. Although isotopic labeling is both expensive and labor intensive, it does yield unambiguous information about both wild-type and mutant proteins. In favorable cases assignments can be made by comparing the NMR data of wild-type and mutant proteins. However, comparative methods are limited to instances of conformationally silent amino acids, or when there is a localized probe of the protein structure.

Original languageEnglish (US)
Pages (from-to)282-292
Number of pages11
JournalMethods in Enzymology
Volume177
Issue numberC
DOIs
StatePublished - Jan 1 1989
Externally publishedYes

Fingerprint

Mutagenesis
Protein Conformation
Mutant Proteins
Site-Directed Mutagenesis
Labeling
Conformations
Hydrogen
Carbon
Amino Acids
Aromatic Amino Acids
Proteins
Nuclear magnetic resonance
Personnel

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Isotopic labeling with hydrogen-2 and carbon-13 to compare conformations of proteins and mutants generated by site-directed mutagenesis, II. / Wilde, Joyce A.; Bolton, Philip H.; Hibler, David W.; Harpold, Lynn; Pourmotabbed, Tayebeh; Dell'Acqua, Mark; Gerlt, John A.

In: Methods in Enzymology, Vol. 177, No. C, 01.01.1989, p. 282-292.

Research output: Contribution to journalArticle

Wilde, Joyce A. ; Bolton, Philip H. ; Hibler, David W. ; Harpold, Lynn ; Pourmotabbed, Tayebeh ; Dell'Acqua, Mark ; Gerlt, John A. / Isotopic labeling with hydrogen-2 and carbon-13 to compare conformations of proteins and mutants generated by site-directed mutagenesis, II. In: Methods in Enzymology. 1989 ; Vol. 177, No. C. pp. 282-292.
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