PubMed · 3374359
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P Monsan, D Combes. 1988. Enzyme stabilization by immobilization.. https://doi.org/10.1016/0076-6879(88)37055-2
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Cross-linking mass spectrometry is a powerful method for structural analysis, but choosing between cleavable and non-cleavable cross-linkers remains challenging. We rigorously compared non-cleavable DSS with cleavable DSSO and found that DSS consistently yields more cross-link identifications from isolated protein complexes to bacterial lysates. The advantage of DSS diminishes as sample complexity increases. At the highest complexity tested-human cell lysate-the trend reverses, with DSSO outperforming DSS. The superior performance of DSS in less complex samples is likely explained by its longer and more flexible spacer arm, which interrogates a spatial volume >40% larger than that of DSSO. For both cross-linkers, the number of identified cross-links decreases as the search space expands, but more steeply for DSS. This sharper decline arises from DSS cross-links producing slightly lower fragment ion coverage, not from the absence of signature ions that could reduce search space. Fragment ion coverage is key to interactome mapping: when coverage reaches 85% or above, identification sensitivity hardly decreases as the search space expands, regardless of the cross-linker used. In summary, we recommend DSS for samples no more complex than bacterial lysates. For interactome mapping of mammalian cells, although DSSO outperforms DSS, neither achieves deep interactome coverage.
Most cellular functions are performed by multi-protein complexes. The identity of the members of such complexes can now be determined by mass spectrometry. Here we show that mass spectrometry can also be used in order to define the spatial organization of these complexes. In this approach, components of a protein complex are purified via molecular interactions using an affinity tagged member and the purified complex is then partially cross-linked. The products are separated by gel electrophoresis and their constituent components identified by mass spectrometry yielding nearest-neighbor relationships. In this study, a member of the yeast nuclear pore complex (Nup85p) was tagged and a six-member sub-complex of the pore was cross-linked and analyzed by 1D SDS-PAGE. Cross-linking reactions were optimized for yield and number of products. Analysis by MALDI mass spectrometry resulted in the identification of protein constituents in the cross-linked bands even at a level of a few hundred femtomoles. Based on these results, a model of the spatial organization of the complex was derived that was later supported by biological experiments. This work demonstrates, that the use of mass spectrometry is the method of choice for analyzing cross-linking experiments aiming on nearest neighbor relationships.
Dynamin is a 100-kDa GTPase with multiple domains. Some of these have known functions, namely, the N-terminal GTPase domain, the PH domain that binds phosphatidylinositol lipids, and the C-terminal proline-arginine-rich domain (PRD) that binds to several SH3 domain-containing dynamin partners. Others, for example, the "middle" located between the GTPase domain and the PH domain and a predicted alpha-helical domain located between the PH domain and PRD, have unknown functions. Dynamin exists as a homotetramer in solution and self-assembles into higher-order structures resembling rings and helical stacks of rings. Dynamin self-assembly stimulates its GTPase activity. We used limited proteolysis to dissect dynamin's domain structure and to gain insight into intradomain interactions that regulate dynamin self-assembly and stimulate GTPase activity. We found that the PH domain functions as a negative regulator of dynamin self-assembly and stimulates GTPase activity and that the alpha-helical domain, termed GED for GTPase effector domain, is required for stimulated GTPase activity.