Photocrosslinking approaches to interactome mapping

Pham, Nam D.; Parker, Randy B.; Kohler, Jennifer J.

doi:10.1016/j.cbpa.2012.10.034

Cited by 125 publications

(118 citation statements)

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“…The abovementioned reactions are specific for one or several amino acids, but more promiscuous reagents also exist, including those with photoreactive groups [38,39]. Expanding the list of reactive residues increases the chances that proteins with fewer acidic or basic residues, such as membrane proteins, can also be crosslinked.…”

Section: Recent Advances In Xl-ms Methodologymentioning

confidence: 99%

“…Zero-length crosslinking, using carbodiimides to bridge amino and carboxyl groups directly without insertion of a spacer, has recently also been applied to larger protein complexes [36,37]. although data analysis in such experiments remains more challenging.The abovementioned reactions are specific for one or several amino acids, but more promiscuous reagents also exist, including those with photoreactive groups [38,39]. Expanding the list of reactive residues increases the chances that proteins with fewer acidic or basic residues, such as membrane proteins, can also be crosslinked.…”

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confidence: 99%

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Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Leitner

Faini

Stengel

et al. 2016

Trends in Biochemical Sciences

348

285

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In recent years, chemical crosslinking of protein complexes and the identification of crosslinked residues by mass spectrometry (XL-MS; sometimes abbreviated as CX-MS) has become an important technique bridging mass spectrometry (MS) and structural biology. By now, XL-MS is well established and supported by publicly available resources as a convenient and versatile part of the structural biologist's toolbox. The combination of XL-MS with cryoelectron microscopy (cryo-EM) and/or integrative modeling is particularly promising to study the topology and structure of large protein assemblies. Among the targets studied so far are proteasomes, ribosomes, polymerases, chromatin remodelers, and photosystem complexes. Here we provide an overview of recent advances in XL-MS, the current state of the field, and a cursory outlook on future challenges. Chemical Crosslinking as a Tool for Structural BiologyStructural biology makes use of many different techniques to elucidate the 3D structures of proteins and protein complexes. While high-resolution structures have traditionally been obtained by X-ray crystallography, cryo-EM is increasingly able to also generate (near) atomic-resolution models. In recent years, techniques and applications of MS have also rapidly progressed. Earlier studies were largely focused on the large-scale identification and quantification of proteins, whereas recent methods also support queries into the composition, stoichiometry, and spatial arrangement of subunits in a complex. These developments have now further progressed toward generating information that contributes, as part of hybrid structural strategies, to the structure elucidation of large molecular assemblies including protein complexes that perform essential processes in the cell. XL-MS is a particularly powerful mass spectrometric technique in this respect, because it provides several layers of information. Identifying protein-protein contacts through XL-MS confirms physical proximity between subunits because the proteins must be close enough in space to be crosslinked. Localizing the side chains that are connected restricts this proximity to certain regions (e.g., domains or even single helices or loops). Finally, the structure of the connected side chains and the crosslinker moiety impart a distance restraint that can be used for molecular modeling purposes because an upper Trends Chemical crosslinking followed by the mass spectrometric analysis of cross linked peptides (XL MS) identifies con tact sites between residues within a single or between multiple proteins.The application of XL MS to many bio logically relevant molecular machines has been shown, with a rapidly growing number of successful studies reported in the past 2 3 years.Crosslinking data are useful in integra tive modeling workflows by providing distance restraints on the surface of folded proteins and complexes. XL MS has been shown to be particularly powerful in combination with 3D cryo electron microscopy. Sciences ; 41 (2016), 1. -S. 20-32 https://dx.doi.org/10.1016...

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Section: Recent Advances In Xl-ms Methodologymentioning

confidence: 99%

mentioning

confidence: 99%

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Leitner

Faini

Stengel

et al. 2016

Trends in Biochemical Sciences

348

285

View full text Add to dashboard Cite

show abstract

“…34 and 35). The photo-reactive amino acid analog pBpa forms cross-links when cells are exposed to UV light (36,37). pBpa is incorporated at a specific location in a bacterial protein by introducing an internal amber codon into the gene and decoding it with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair FIGURE 4.…”

Section: Detection Of a Contact Between Pspa And The C-terminal Domaimentioning

confidence: 99%

Activity of a Bacterial Cell Envelope Stress Response Is Controlled by the Interaction of a Protein Binding Domain with Different Partners

Flores-Kim

Darwin

2015

Journal of Biological Chemistry

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“…Cysteine-specific cross-linking is also well established but usually does not yield sufficient structural information due to the low prevalence of cysteines in proteins and their involvement in the formation of disulfide bonds. Zero-length cross-linking by carbodiimide coupling (17)(18)(19) and photochemical cross-linking (20) are other strategies that have been described but have not yet found widespread application in the field.…”

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confidence: 99%

Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

Leitner

Joachimiak

Unverdorben

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

226

312

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The study of proteins and protein complexes using chemical crosslinking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. P roteins exert the majority of their functions in the form of protein complexes to control cellular signaling, protein synthesis, folding and degradation, and many more essential processes. Therefore, elucidating the composition and structure of such complexes has been a longstanding goal of biological research.MS-based proteomics has emerged as one of the main techniques to identify and quantify proteins and their modifications in biological samples such as isolated complexes, proteome fractions, or whole proteomes. Various MS methods now provide structural information on protein assemblies (1-3). Among them, chemical cross-linking and identification of cross-linked peptides by MS (XL-MS) has been increasingly applied to determine the subunit arrangements of biologically relevant complexes (4-6). Such XL-MS experiments indicate the locations of cross-linking sites and thus the spatial proximity of reactive groups that are connected by a covalent bond. This information is then used to determine the positioning of subunits or locate interacting regions, alone or in combination with other techniques such as NMR spectroscopy, electron microscopy, and X-ray crystallography.In the last few years, optimized protocols and new computational tools for the reliable analysis of XL-MS datasets resulted in significant advances of the XL-MS technology (4-6). These advances have contributed to the emergence of a robust, integrated XL-MS method that has been successfully applied for structure determination of a number of large protein complexes (7-11) and the detection of direct, physical interactions in whole cells (12)(13)(14). To date, the cross-linking chemistries applied in these studies have targ...

show abstract

Photocrosslinking approaches to interactome mapping

Cited by 125 publications

References 70 publications

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Activity of a Bacterial Cell Envelope Stress Response Is Controlled by the Interaction of a Protein Binding Domain with Different Partners

Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

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