Topographic Studies of the GroEL-GroES Chaperonin Complex by Chemical Cross-linking Using Diformyl Ethynylbenzene

Abstract

Many essential cellular processes depend upon the selfassembly of stable multiprotein entities. The architectures of the vast majority of these protein machines remain unknown because these structures are difficult to obtain by biophysical techniques alone. However, recent progress in defining the architecture of protein complexes has resulted from integrating information from all available biochemical and biophysical sources to generate computational models. Chemical cross-linking is a technique that holds exceptional promise toward achieving this goal by providing distance constraints that reflect the topography of protein complexes. Combined with the available structural data, these constraints can yield three-dimensional models of higher order molecular machines. However, thus far the utility of cross-linking has been thwarted by insufficient yields of cross-linked products and tandem mass spectrometry methods that are unable to unambiguously establish the identity of the covalently labeled peptides and their sites of modification. We report the cross-linking of amino moieties by 1,3-diformyl-5-ethynylbenzene (DEB) with analysis by high resolution electron transfer dissociation. This new reagent coupled with this new energy deposition technique addresses these obstacles by generating cross-linked peptides containing two additional sites of protonation relative to conventional cross-linking reagents. In addition to excellent coverage of sequence ions by electron transfer dissociation, DEB crosslinking produces gas-phase precursor ions in the 4؉, 5؉, or 6؉ charge states that are readily segregated from unmodified and dead-end modified peptides using charge-dependent precursor selection of only quadruply and higher charge state ions. Furthermore, electron transfer induces dissociation of the DEB-peptide bonds to yield diagnostic ion signals that reveal the "molecular ions" of the unmodified peptides. We demonstrate the power of this strategy by cross-linking analysis of the 21-protein, ADP-bound GroELGroES chaperonin complex. Twenty-five unique sites of cross-linking were determined. Molecular & Cellular Proteomics 9:2306 -2317, 2010.A wide range of cellular processes are mediated by stable protein complexes that range in subunit size from a few proteins, e.g. the signal recognition particle, to over a hundred, e.g. the spliceosome. Indeed, protein interactions underlie an enormous scope of physiological and pathophysiological processes, encompassing everything from cell cycle regulation to initiation of apoptosis, angiogenesis, and aberrant interactions in cancer.Presently, the subunit compositions, dynamics, and topographies as well as the overall architectures of most multimeric complexes remain unknown. With a handful of exceptions, most notably the crystal structures of the large and small ribosomal subunits, which were early synchrotron successes determined some 10 years ago (1-3), large molecular machines have proven recalcitrant to high resolution structural analysis. Conversely, a large number of indiv...

Matching Cross-linked Peptide Spectra: Only as Good as the Worse Identification

“…In this respect, the use of electron transfer dissociation fragmentation instead of collision-induced dissociation could be important, and the development of new cross-linkers that benefit electron transfer dissociation performance may have an impact (41). The use of MS-cleavable cross-linkers should also better guarantee fragments from both peptides in MS3 spectra (14 -18).…”

Cross-Link Guided Molecular Modeling with ROSETTA

“…Although XL-MS only provides comparatively low resolution data [6], [7], it is highly complementary to conventional methods, has less stringent sample purity requirements and few restrictions concerning complex size. Recent applications of XL-MS have elucidated the structure and topology of RNA polymerases [8], [9], proteasomes [10], [11] and the chaperonins GroEL [12] and TRiC [13], [14]. Most often, homobifunctional cross-linking reagents, for example amine-reactive succinimide esters are used in XL-MS studies.…”

Protein Structural Analysis via Mass Spectrometry-Based Proteomics

“…CXMS provides a range of structural information, and the resolution of this information is dependent on how specifically a crosslinking (CX) site can be localized in the context of a protein target, with the identification of crosslinked amino acid side chains providing the highest structural resolution. CX sites may be used to determine the proximity of domains and amino acid side chains in protein monomers or complexes, to identify potential intramolecular or intermolecular protein binding sites, and to provide structural constraints for theoretical protein models (89–91). Many search algorithms and specialized reagents have been developed to enrich and enhance the detection of conjugates and more numerous side products in digests of crosslinked proteins (90,92–94), making this approach readily accessible to any researcher with access to MS and proteomics facilities.…”

“…Several groups have developed CXRs that fragment during MS/MS to release small molecules that provide mass signatures for crosslinked peptides (95), termed protein interaction reporters (102). Using a different ligation approach, Trnka and Burlingame synthesized a novel CXR, diformyl ethynlbenzene (DEB), which forms Schiff bases with lysine ε-amines that are subsequently reduced to secondary amines with cyanoborohydride (91). The authors demonstrate that reduction to the amine, rather than the common acetylation product formed by NHS groups, provides two additional protonation centers with incorporation of the intervening rigid ring spacer, decreasing the m/z ratio of the conjugate for more optimal fragmentation by high resolution ETD and electron capture dissociation (ECD) fragmentation methods, which provide more complete fragmentation along the peptide backbone.…”

“…Moreover, the reagent contains a clickable tag for purification and fragments during MS/MS to produce diagnostic reporter groups. Digestion of DEB crosslinked proteins also generates high charge state gas phase precursor ions (4–6 + ), which allows for their exclusion from native and dead-end modified peptide ions using charge dependent precursor selection (91). More recently Ihling et al .…”