Abstract
Multi-signal sedimentation velocity analytical ultracentrifugation (MSSV) is a powerful tool for the determination of the number, stoichiometry, and hydrodynamic shape of reversible protein complexes in two- and three-component systems. In this method, the evolution of sedimentation profiles of macromolecular mixtures is recorded simultaneously using multiple absorbance and refractive index signals and globally transformed into both spectrally and diffusion-deconvoluted component sedimentation coefficient distributions. For reactions with complex lifetimes comparable to the time-scale of sedimentation, MSSV reveals the number and stoichiometry of co-existing complexes. For systems with short complex lifetimes, MSSV reveals the composition of the reaction boundary of the coupled reaction/migration process, which we show here may be used to directly determine an association constant. A prerequisite for MSSV is that the interacting components are spectrally distinguishable, which may be a result, for example, of extrinsic chromophores or of different abundances of aromatic amino acids contributing to the UV absorbance. For interacting components that are spectrally poorly resolved, here we introduce a method for additional regularization of the spectral deconvolution by exploiting approximate knowledge of the total loading concentrations. While this novel mass conservation principle does not discriminate contributions to different species, it can be effectively combined with constraints in the sedimentation coefficient range of uncomplexed species. We show in theory, computer simulations, and experiment, how mass conservation MSSV as implemented in SEDPHAT can enhance or even substitute for the spectral discrimination of components. This should broaden the applicability of MSSV to the analysis of the composition of reversible macromolecular complexes.
Highlights
The study of protein interactions in multi-component systems is key to improve our understanding of signaling pathways, which ubiquitously possess dynamically assembled multi-protein complexes as critical nodes for integrating different information flows and regulating downstream events
Computer simulations As a first test of the MC-multi-signal sedimentation velocity (MSSV) analysis approach, sedimentation profiles were simulated for a 100 kg/mol, 6S-protein ‘B’ binding a 20 kg/mol, 2 S-protein ‘A’ with different absorbance extinction coefficients e280 = 20,630, 23,180, or 26,500 M21 cm21 corresponding to the different Dnorm values 0.01, 0.05 and 0.10, respectively, creating a 7 S complex with Kd of 2 mM and koff = 1022/sec (System 1)
To assess how well the molar ratio can be defined by the data, we probed the change in the quality of fit that occurred when the Ck(s) distribution in the range of the sedimentation coefficient of the complex was described by only a single class of species of a composition pre-constrained to different molar ratio values (Fig. 1)
Summary
The study of protein interactions in multi-component systems is key to improve our understanding of signaling pathways, which ubiquitously possess dynamically assembled multi-protein complexes as critical nodes for integrating different information flows and regulating downstream events. Hallmarks of such complexes are multi-valent interactions and cooperativity, which are notoriously difficult to characterize. The multi-signal sedimentation velocity (MSSV) approach was introduced [2] as a new tool to address this problem It takes advantage of the strongly size-dependent migration in the centrifugal field in a configuration that leaves complexes always in a bath of their components, such as to maintain populated complexes in solution during the experiment despite their differential sedimentation velocities. Many applications of MSSV to two- and threecomponent systems have demonstrated the power of this approach [6,7,8,9,10,11,12,13,14,15,16]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
