New chemistry for the study of multiprotein complexes: the six-histidine tag as a receptor for a protein crosslinking 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 highly purified protein, limiting the general utility of these approaches. The need for covalent posttranslational chemistry also makes it extremely difficult to use modified proteins in studies of native complexes in crude lysates or in living cells. We set out to develop a protein tag that would circumvent these problems.


RESULTS
A very simple type of molecular recognition, metal-ligand complexation, can be used to deliver a nickel-based crosslinking reagent to proteins containing a six-histidine (His6) tag. When activated with a peracid, the His6-Ni complex mediates oxidative crosslinking of nearby proteins. The crosslinking reaction does not involve freely diffusible intermediates, and thus only those proteins in close proximity to the His6-tagged polypeptide are crosslinked.


CONCLUSIONS
The His6 tag, commonly used as an affinity handle for the purification of recombinant proteins, can also be used as an internal receptor for an oxidative protein-crosslinking reagent. No covalent protein modifications are necessary, since the His6 tag is introduced at the DNA level. The crosslinking reaction is fast, efficient in most cases, and provides products that are easily separated from most other proteins present. This methodology should find widespread use in the study of multiprotein complexes.
Fancy
D A
DA
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX 78712-1096, USA. kodadek@ccwf.cc.texas.edu
Melcher
K
K
Johnston
S A
SA
Kodadek
T
T
D001665:Binding Sites; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008024:Ligands; D046911:Macromolecular Substances; D008958:Models, Molecular; D046912:Multiprotein Complexes; D010084:Oxidation-Reduction; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry*; D051135:Rad51 Recombinase; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008024:Ligands; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D011506:Proteins; D011994:Recombinant Proteins; D006639:Histidine; D051135:Rad51 Recombinase