Chemical determination of protein neighbourhoods in a cellular organelle

Experimental procedures developed for the identification of near-neighboring proteins in a complicated organelle are described in this paper. The specific system studied here is the ribosome of Escherichia coli; however, the techniques and reagents described should have a wide range of applications. We have used three kinds of cross-linking agents: noncleavable maleimide reagents which react with free SH groups; noncleavable diimidoesters which react with free amino groups, and cleavable diazide agents that also react with free amino groups. The combination of immunological and isotopic labelling techniques used to identify proteins cross-linked by noncleavable reagents, as well as the special electrophoretic techniques used with the cleavable reagents are described. We show that the neighborhoods detected by the crosslink technology are functionally meaningful ones, and also discuss the ambiguities inherent in the structural interpretation of such protein neighborhoods.