1. An integrated chemical cross-linking and mass spectrometry approach to study protein complex architecture and function
Jie Luo, James Fishburn, Steven Hahn, Jeffrey Ranish Mol Cell Proteomics. 2012 Feb;11(2):M111.008318. doi: 10.1074/mcp.M111.008318. Epub 2011 Nov 7.
Knowledge of protein structures and protein-protein interactions is essential for understanding biological processes. Chemical cross-linking combined with mass spectrometry is an attractive approach for studying protein-protein interactions and protein structure, but to date its use has been limited largely by low yields of informative cross-links (because of inefficient cross-linking reactions) and by the difficulty of confidently identifying the sequences of cross-linked peptide pairs from their fragmentation spectra. Here we present an approach based on a new MS labile cross-linking reagent, BDRG (biotin-aspartate-Rink-glycine), which addresses these issues. BDRG incorporates a biotin handle (for enrichment of cross-linked peptides prior to MS analysis), two pentafluorophenyl ester groups that react with peptide amines, and a labile Rink-based bond between the pentafluorophenyl groups that allows cross-linked peptides to be separated during MS and confidently identified by database searching of their fragmentation spectra. We developed a protocol for the identification of BDRG cross-linked peptides derived from purified or partially purified protein complexes, including software to aid in the identification of different classes of cross-linker-modified peptides. Importantly, our approach permits the use of high accuracy precursor mass measurements to verify the database search results. We demonstrate the utility of the approach by applying it to purified yeast TFIIE, a heterodimeric transcription factor complex, and to a single-step affinity-purified preparation of the 12-subunit RNA polymerase II complex. The results show that the method is effective at identifying cross-linked peptides derived from purified and partially purified protein complexes and provides complementary information to that from other structural approaches. As such, it is an attractive approach to study the topology of protein complexes.
2. Synthesis of hetero-telechelic alpha,omega bio-functionalized polymers
Peter J Roth, Florian D Jochum, Rudolf Zentel, Patrick Theato Biomacromolecules. 2010 Jan 11;11(1):238-44. doi: 10.1021/bm901095j.
Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize poly[diethylene glycol monomethylether methacrylate] (PDEGMA) (M(n) = 6250 g/mol, PDI = 1.14) with a pentafluorophenyl (PFP) activated ester and a dithioester end group. The hormone thyroxin (T4) was quantitatively attached to the PFP activated ester alpha end group via its amino group. The omega-terminal dithioester was not harmed by this reaction and was subsequently aminolyzed in the presence of N-biotinylaminoethyl methanethiosulfonate, yielding a polymer with a thyroxin and a biotin end group with very high heterotelechelic functionality. The polymer was characterized by (1)H, (13)C, and (19)F NMR, UV-vis, and IR spectroscopy and gel permeation chromatography. The thyroxin transport protein prealbumin with two thyroxin binding sites and streptavidin, which has four biotin binding sites, was conjugated using the biotarget labeled polymer, resulting in the formation of a protein-polymer network, confirming the heterotelechelic nature of the polymer. Polymer-protein microgel formation was observed with dynamic light scattering. To realize a directed protein assembly, prealbumin was immobilized onto a surface, exposing one of its two thyroxin binding groups and thus allowing the conjugation with the thyroxin alpha end group of the heterotelechelic polymer. The biotin omega end group of the attached polymer layer enabled the subsequent immobilization of streptavidin, yielding a defined multilayer system of two proteins connected with the synthetic polymer (efficiency of streptavidin immobilization 81% based on prealbumin). Without the polymer, no streptavidin immobilization occurred. The layer depositions were monitored by surface plasmon resonance. The synthetic approach of combining PFP activated esters with functional MTS reagents presents a powerful method for obtaining well-defined heterotelechelic (bio-) functionalized polymers.
3. Bifunctional activity labels for selection of filamentous bacteriophages displaying enzymes
S Vanwetswinkel, R Touillaux, J Fastrez, J Marchand-Brynaert Bioorg Med Chem. 1995 Jul;3(7):907-15. doi: 10.1016/0968-0896(95)00084-t.
Two bifunctional activity labels of beta-lactamases or penicillin binding proteins have been prepared. They feature a penicillin sulfone derivative, i.e. a suicide substrate of serine beta-lactamases, or a penicillin derivative connected to a biotin moiety through a spacer containing a disulfide bridge. The biotinyl spacer 4 was prepared by coupling biotin to epsilon-amino-caproic acid, then to cystamine, and purified by transient protection with t-Boc. The penicillin sulfone inhibitor 13 was prepared by chemoselective sulfonylation of methoxymethyl 6-aminopenicillinate with pentafluorophenoxy- or benzyloxy-carbonylmethylsulfonyl chloride (9), followed by permanganate oxidation. Both direct coupling of the activated ester 13b and indirect coupling of the acid 13c obtained by benzyl ester deprotection, afforded the biotinylated sulfone inhibitor 16. The acid 6 resulting from reaction of the biotinyl spacer 4 with glutaric anhydride was activated as pentafluorophenyl-ester 7 and reacted with 6-aminopenicillanic acid to afford the penicillin binding protein label 18. Selection of the most active beta-lactamase displayed on phage from a mixture containing less active enzymes could be accomplished in three rounds of labeling and affinity chromatography using suicide inhibitor 16.
