Two-dimensional NMR as a probe of structural similarity applied to mutants of cytochrome c.
Using site-directed mutagenesis, it is possible to prepare many mutants of a protein in a short time, and to uncover differences in function. To understand the changes in function, it is essential to understand the effect(s) of the mutation in terms of structural and dynamic changes. It is particularly important to establish a rapid method for comparing the structure of the mutants with that of the wild-type protein. We propose that a combination of overlayed and difference two-dimensional NOE spectra between the wild-type and mutant protein provide a rapid method for determination of structural similarity. The observation of differences other than those due directly to the field effects of the exchanged side chain allow both local and distant conformational changes to be assessed. Here we compare NOESY spectra from a mutant of yeast iso-1-ferrocytochrome c in which the invariant residue Phe-82 has been changed to a Tyr. We conclude that NMR can show subtle changes in protein structure. Specifically, we show the change must involve the reorientation of the side chain of Leu-85 which is proximal to the mutation. The dynamics of the aromatic side chain at position 82 are shown not to give rise to measurable differences between the wild-type and mutant protein. Structural changes are not propagated to a measurable degree in other parts of the protein.
Pielak
G J
GJ
Inorganic Chemistry Laboratory, University of Oxford, England.
Atkinson
R A
RA
Boyd
J
J
Williams
R J
RJ
D001665:Binding Sites; D003574:Cytochrome c Group / Q000032:analysis / Q000235:genetics*; D045304:Cytochromes c; D004735:Energy Transfer; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011487:Protein Conformation; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D015003:Yeasts / Q000201:enzymology
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c