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Subunit pI Can Influence Protein Complex Dissociation Characteristics.

ABSTRACT: Mass spectrometry is frequently used to determine protein complex topology. By combining in-solution and gas-phase dissociation measurements, information can be indirectly inferred about the original composition of the protein complex. Although the mechanisms behind gas-phase complex dissociation are becoming more established, protein complex dissociation is not always predictable. Here, we looked into the effect of the protein subunits pI on complex dissociation. We chose two structurally similar, hexameric protein complexes that consist of a ring of alternating alpha and beta subunits. For one complex, allophycocyanin, the alpha and beta subunits are structurally similar, almost identical in mass, but have distinct pIs. In contrast, the other complex, phycoerythrin, is structural similar to allophycocyanin, yet the subunits have identical pIs. As predicted based on the structural arrangement, dissociation of phycoerythrin resulted in the observation of both the alpha and beta monomeric subunits in the mass spectrometer. However, for allophycocyanin, the results differed dramatically, with only the alpha monomeric subunit being detected upon gas-phase dissociation. Together, the results highlighted the importance of considering the isoelectric points of individual subunits within a protein complex when using tandem mass spectrometry data to elucidate protein complex topology.

SUBMITTER: Leney AC

PROVIDER: S-EPMC6669198 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Subunit pI Can Influence Protein Complex Dissociation Characteristics.

Leney Aneika C AC

Journal of the American Society for Mass Spectrometry 20190510 8

Mass spectrometry is frequently used to determine protein complex topology. By combining in-solution and gas-phase dissociation measurements, information can be indirectly inferred about the original composition of the protein complex. Although the mechanisms behind gas-phase complex dissociation are becoming more established, protein complex dissociation is not always predictable. Here, we looked into the effect of the protein subunits pI on complex dissociation. We chose two structurally simil ...[more]

PMID: 31077092

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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.

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Subunit pI Can Influence Protein Complex Dissociation Characteristics.

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Subunit pI Can Influence Protein Complex Dissociation Characteristics.

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