Chemistry for the analysis of protein–protein interactions: Rapid and efficient cross-linking triggered by long wavelength light

Abstract

Chemical cross-linking is a potentially useful technique for probing the architecture of multiprotein complexes. However, analyses using typical bifunctional cross-linkers often suffer from poor yields, and large-scale modification of nucleophilic side chains can result in artifactual results attributable to structural destabilization. We report here the de novo design and development of a type of protein cross-linking reaction that uses a photogenerated oxidant to mediate rapid and efficient cross-linking of associated proteins. The process involves brief photolysis of tris-bipyridylruthenium(II) dication with visible light in the presence of the electron acceptor ammonium persulfate and the proteins of interest. Very high yields of cross-linked products can be obtained with irradiation times of <1 second. This chemistry obviates many of the problems associated with standard cross-linking reagents.

Oligomerization state and supramolecular structure of the HIV‐1 Vpu protein transmembrane segment in phospholipid bilayers

“…To probe the oligomeric state of Vpu in lipid bilayers, we used the PICUP technique 48,49 in analogy to experiments by Bitan and Teplow in which PICUP has been used to characterize oligomer size distributions in solutions of amyloid-forming peptides. 50,51 Bilayers for PICUP experiments and solid-state NMR experiments were comprised of DOPC and DOPG, in a 9:1 molar ratio.…”

“…49 Y29, the only tyrosine residue in Vpu , is located outside the TM segment and is presumably in the aqueous phase, where it would be readily accessible to oxidation. Therefore, we examined Y29T-Vpu 1-40 to see whether this mutant might be resistant to crosslinking.…”

“…[48][49][50] For these experiments, Vpu 1-40 was dissolved in trifluoroethanol and then mixed with DOPC/DOPG (9:1) in chloroform, producing a 0.1-3.0% mole fraction of peptide relative to phospholipids. The mixture was dried under nitrogen gas and left in vacuum for 4 h, then hydrated using 10 mM sodium phosphate buffer (pH 7.4) to produce a final Vpu 1-40 concentration of 20 lM for a 0.3% mole fraction.…”

Use of synthetic signal sequences to explore the protein export machinery

“…We used benzophenone-mediated (BPM) crosslinking 61 and photoinduced crosslinking of unmodified proteins (PICUP). 62,63 Here, both methods lead to a covalent linkage between the SRP (in vitro reconstituted using recombinant Ffh protein from E. coli and a 43-oligonucleotide RNA, representing the minimal element of 4.5S RNA, transcribed in vitro) and solid-phase synthesized variants of a Leu-enriched alkaline phosphatase signal sequencebased peptide (PhoA; see Table I). It had been shown previously in an in vitro translation system that these signal sequences can interact strongly with E. coli SRP.…”

“…We speculate that the interactions between the signal peptide and the NG domain can be captured using PICUP because of the spatial requirements of the reaction: only short-distance interaction between the side chains of interacting peptides/proteins that are close to each other can be observed by this method. 62,63 Also, crosslinking by PICUP only happens between groups with the appropriate reactivity, and the reaction requires very short time ( 1 s). Our observations are consistent with a model in which the NG domain interacts closely with the signal peptide, at least part of the time, emphasizing the extensive dynamics of the recognition process.…”