Affinity immobilisation and "negative" crosslinking: a probe for tertiary and quaternary protein structure

Abstract

Limited structural information has hitherto been obtainable from crosslinking studies on purified proteins. A major limitation has been the lack of a procedure permitting confident interpretation of "negative" crosslinking data. The "spreading-out" of a protein on an affinity matrix at a critically low density below which intermolecular bridge formation does not occur, prior to reaction with a chemical crosslinker (which may be safely added in high concentrations), provides an approach which yielded valuable information regarding the tertiary and quaternary structure of concanavalin A. This information could be cross-checked from the existing data on this protein previously obtained by crystallography and other biophysical techniques, thus demonstrating the validity of this probe. This matrix approach promises to be particularly useful in the structural study of poorly soluble membrane-bound proteins and other proteins which are difficult to crystallise.