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Journal article
Multi-Signal Sedimentation Velocity Analysis with Mass Conservation for Determining the Stoichiometry of Protein Complexes
PloS one, v 8(5), pp e62694-e62694
16 May 2013
PMID: 23696787
Featured in Collection : UN Sustainable Development Goals @ Drexel
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.
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Details
- Title
- Multi-Signal Sedimentation Velocity Analysis with Mass Conservation for Determining the Stoichiometry of Protein Complexes
- Creators
- Chad A. Brautigam - College Station Medical CenterShae B. Padrick - College Station Medical CenterPeter Schuck - Natl Inst Biomed Imaging & Bioengn, Lab Cellular Imaging & Macromol Biophys, NIH, Bethesda, MD USA
- Publication Details
- PloS one, v 8(5), pp e62694-e62694
- Publisher
- Public Library Science
- Number of pages
- 14
- Grant note
- ZIAEB000051 / NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Biomedical Imaging & Bioengineering (NIBIB) National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, DHHS
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology
- Web of Science ID
- WOS:000319081900004
- Scopus ID
- 2-s2.0-84877793564
- Other Identifier
- 991020836483904721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemistry & Molecular Biology