NEW CHEMISTRY FOR THE STUDY OF MULTIPROTEIN COMPLEXES - THE 6-HISTIDINE TAG AS A RECEPTOR FOR A PROTEIN CROSS-LINKING REAGENT

Background: To study very large macromolecular complexes, it would be useful to be able to incorporate probe molecules, such as fluorescent tags or photoactivatable crosslinkers, into specific sites on proteins . Current methods for doing this use relatively large amounts of highl y purified protein, limiting the general utility of these approaches. The need for covalent posttranslational chemistry also makes it extrem ely difficult to use modified proteins in studies of native complexes in crude lysates or in living cells. We set out to develop a protein t ag that would circumvent these problems. Results: A very simple type o f molecular recognition, metal-ligand complexation, can be used to del iver a nickel-based crosslinking reagent to proteins containing a six- histidine (His(6)) tag. When activated with a peracid, the His(6)-Ni c omplex mediates oxidative crosslinking of nearby proteins. The crossli nking reaction does not involve freely diffusible intermediates, and t hus only those proteins in close proximity to the His(6)-tagged polype ptide are crosslinked. Conclusions: The His(6) tag, commonly used as a n affinity handle for the purification of recombinant proteins, can al so be used as an internal receptor for an oxidative protein-crosslinki ng reagent. No covalent protein modifications are necessary, since the His(6) tag is introduced at the DNA level. The crosslinking reaction is fast, efficient in most cases, and provides products that are easil y separated from most other proteins present. This methodology should find widespread use in the study of multiprotein complexes.