Abstract:
:Sedimentation velocity analytical ultracentrifugation with fluorescence detection has emerged as a powerful method for the study of interacting systems of macromolecules. It combines picomolar sensitivity with high hydrodynamic resolution, and can be carried out with photoswitchable fluorophores for multicomponent discrimination, to determine the stoichiometry, affinity, and shape of macromolecular complexes with dissociation equilibrium constants from picomolar to micromolar. A popular approach for data interpretation is the determination of the binding affinity by isotherms of weight-average sedimentation coefficients sw. A prevailing dogma in sedimentation analysis is that the weight-average sedimentation coefficient from the transport method corresponds to the signal- and population-weighted average of all species. We show that this does not always hold true for systems that exhibit significant signal changes with complex formation-properties that may be readily encountered in practice, e.g., from a change in fluorescence quantum yield. Coupled transport in the reaction boundary of rapidly reversible systems can make significant contributions to the observed migration in a way that cannot be accounted for in the standard population-based average. Effective particle theory provides a simple physical picture for the reaction-coupled migration process. On this basis, we develop a more general binding model that converges to the well-known form of sw with constant signals, but can account simultaneously for hydrodynamic cotransport in the presence of changes in fluorescence quantum yield. We believe this will be useful when studying interacting systems exhibiting fluorescence quenching, enhancement, or Förster resonance energy transfer with transport methods.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Chaturvedi SK,Zhao H,Schuck Pdoi
10.1016/j.bpj.2017.02.020subject
Has Abstractpub_date
2017-04-11 00:00:00pages
1374-1382issue
7eissn
0006-3495issn
1542-0086pii
S0006-3495(17)30234-5journal_volume
112pub_type
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