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Resolution of a protein sequence ambiguity by X‐ray crystallographic and mass spectrometric methods
Author(s) -
Keefe L. J.,
Lattman E. E.,
Wolkow C.,
Woods A.,
Chevrier M.,
Cotter R. J.
Publication year - 1992
Publication title -
journal of applied crystallography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.429
H-Index - 162
ISSN - 1600-5767
DOI - 10.1107/s0021889891011986
Subject(s) - mass spectrometry , chemistry , crystallography , crystal structure , peptide sequence , resolution (logic) , amino acid , alanine , residue (chemistry) , protein crystallization , sequence (biology) , protein structure , glycine , crystallization , biochemistry , chromatography , artificial intelligence , computer science , gene , organic chemistry
Ambiguities in amino acid sequences are a potential problem in X‐ray crystallographic studies of proteins. Amino acid side chains often cannot be reliably identified from the electron density. Many protein crystal structures that are now being solved are simple variants of a known wild‐type structure. Thus, cloning artifacts or other untoward events can readily lead to cases in which the proposed sequence is not correct. An example is presented showing that mass spectrometry provides an excellent tool for analyzing suspected errors. The X‐ray crystal structure of an insertion mutant of Staphylococcal nuclease has been solved to 1.67 Å resolution and refined to a crystallographic R value of 0.170 [Keefe & Lattman (1992). In preparation]. A single residue has been inserted in the C‐terminal α helix. The inserted amino acid was believed to be an alanine residue, but the final electron density maps strongly indicated that a glycine had been inserted instead. To confirm the observations from the X‐ray data, matrix‐assisted laser desorption mass spectrometry was employed to verify the glycine insertion. This mass spectrometric technique has sufficient mass accuracy to detect the methyl group that distinguishes glycine from alanine and can be extended to the more common situation in which crystallographic measurements suggest a problem with the sequence, but cannot pinpoint its location or nature.