Dataset Information

Revealing the quaternary structure of a heterogeneous noncovalent protein complex through surface-induced dissociation.

ABSTRACT: As scientists begin to appreciate the extent to which quaternary structure facilitates protein function, determination of the subunit arrangement within noncovalent protein complexes is increasingly important. While native mass spectrometry shows promise for the study of noncovalent complexes, few developments have been made toward the determination of subunit architecture, and no mass spectrometry activation method yields complete topology information. Here, we illustrate the surface-induced dissociation of a heterohexamer, toyocamycin nitrile hydratase, directly into its constituent trimers. We propose that the single-step nature of this activation in combination with high energy deposition allows for dissociation prior to significant unfolding or other large-scale rearrangement. This method can potentially allow for dissociation of a protein complex into subcomplexes, facilitating the mapping of subunit contacts and thus determination of quaternary structure of protein complexes.

SUBMITTER: Blackwell AE

PROVIDER: S-EPMC3343771 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Revealing the quaternary structure of a heterogeneous noncovalent protein complex through surface-induced dissociation.

Blackwell Anne E AE Dodds Eric D ED Bandarian Vahe V Wysocki Vicki H VH

Analytical chemistry 20110321 8

As scientists begin to appreciate the extent to which quaternary structure facilitates protein function, determination of the subunit arrangement within noncovalent protein complexes is increasingly important. While native mass spectrometry shows promise for the study of noncovalent complexes, few developments have been made toward the determination of subunit architecture, and no mass spectrometry activation method yields complete topology information. Here, we illustrate the surface-induced di ...[more]

PMID: 21417466

Similar Datasets

Project description:Recently, mass spectrometry (MS) has become a viable method for elucidation of protein structure. Surface-induced dissociation (SID), colliding multiply charged protein complexes or other ions with a surface, has been paired with native MS to provide useful structural information such as connectivity and topology for many different protein complexes. We recently showed that SID gives information not only on connectivity and topology but also on relative interface strengths. However, SID has not yet been coupled with computational structure prediction methods that could use the sparse information from SID to improve the prediction of quaternary structures, i.e., how protein subunits interact with each other to form complexes. Protein-protein docking, a computational method to predict the quaternary structure of protein complexes, can be used in combination with subunit structures from X-ray crystallography and NMR in situations where it is difficult to obtain an experimental structure of an entire complex. While de novo structure prediction can be successful, many studies have shown that inclusion of experimental data can greatly increase prediction accuracy. In this study, we show that the appearance energy (AE, defined as 10% fragmentation) extracted from SID can be used in combination with Rosetta to successfully evaluate protein-protein docking poses. We developed an improved model to predict measured SID AEs and incorporated this model into a scoring function that combines the RosettaDock scoring function with a novel SID scoring term, which quantifies agreement between experiments and structures generated from RosettaDock. As a proof of principle, we tested the effectiveness of these restraints on 57 systems using ideal SID AE data (AE determined from crystal structures using the predictive model). When theoretical AEs were used, the RMSD of the selected structure improved or stayed the same in 95% of cases. When experimental SID data were incorporated on a different set of systems, the method predicted near-native structures (less than 2 Å root-mean-square deviation, RMSD, from native) for 6/9 tested cases, while unrestrained RosettaDock (without SID data) only predicted 3/9 such cases. Score versus RMSD funnel profiles were also improved when SID data were included. Additionally, we developed a confidence measure to evaluate predicted model quality in the absence of a crystal structure.

Project description:Herein, we focus on native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), and illustrate its utility in the characterization of protein complex topology and glycoprotein heterogeneity. ECCR is illustrated to effectively spread the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurement out to greater than 100,000 m/z. For both the pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) lowers the charge states and produces more topologically informative fragmentation. While more native-like fragmentation has previously been illustrated for complexes charge reduced by proton abstraction in solution, this is the first illustration that ECCR can lead to more native-like SID fragmentation of protein complexes. Application to protein glycosylation, one of the most common and diverse protein posttranslational modifications, is also illustrated because glycosylation is important for structural and functional properties and plays essential roles in many key biological processes. The immense heterogeneity resulting from variability in glycosylation sites and glycan composition and structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Data for stabilized heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer illustrate that ECCR enables significantly improved resolution of glycan heterogeneity. Without ECCR, the charge states of a glycoprotein complex are not resolved and average mass determination is available only through the use of charge detection mass spectrometry or mass photometry. With ECCR after narrow m/z selection, multiple glycoform m/z values are apparent, providing quick global, glycoform profiling and providing a future path for glycan localization on individual intact glycoforms (e.g., though top-down dissociation).

Dataset Information

Revealing the quaternary structure of a heterogeneous noncovalent protein complex through surface-induced dissociation.

Publications

Revealing the quaternary structure of a heterogeneous noncovalent protein complex through surface-induced dissociation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets