Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield

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 s. 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 s 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.

Nucleic acid–induced dimerization of HIV-1 Gag protein

“…All species' extinction coefficients (or signal coefficients) are denoted by . As we have shown previously (36), the distinction between contributions from undisturbed and reaction boundary is key for studying systems with rapidly interconverting complex formation associated with signal changes such as fluorescence quenching. (It reduces to the conventional signal-weighted average over all species in the absence of signal changes (36).)…”

“…As we have shown previously (36), the distinction between contributions from undisturbed and reaction boundary is key for studying systems with rapidly interconverting complex formation associated with signal changes such as fluorescence quenching. (It reduces to the conventional signal-weighted average over all species in the absence of signal changes (36).) For example, for low protein concentrations, Equation 7 reduces to the following, where c N,u is the fraction of free oligonucleotide remaining in the undisturbed boundary, and c N,r is the corresponding fraction co-transported in the reaction boundary.…”

“…An unusual aspect of the sedimentation analysis is that both the quenching isotherms of Fig. 6 and the s w isotherm need to be analyzed jointly: As pointed out recently, accounting for signal quenching in different states is a critical element for the interpretation of the average signal transport in sedimentation (36). Conversely, the sedimentation velocity information links quenching states to oligomeric states.…”

“…Although titrations of fluorescence quenching can be carried out in bench-top spectrofluorometers, their interpretation is significantly enhanced when the microscopic information from quenching can be combined with size information of macroscopic states observed through hydrodynamics in SV, so as to clarify the binding mechanism and the complex state associated with a quenching state. Conversely, the interpretation of the sedimentation signals is not possible without accounting for fluorescence signal changes in the different states (36). We believe that the combination of high-resolution hydrodynamics with fluorescence intensity quenching, energy transfer, or other proximity-based signals will be similarly applicable and provide a useful tool in the study of other complex multiprotein interactions.…”

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies

“…Moreover, high hydrodynamic resolution of AUC coupled with high-selectivity fluorescent detection can yield more reliable results compared with traditional methods for measuring high-affinity interactions, such as surface plasmon resonance and isothermal titration calorimetry (Chaturvedi et al 2017a). A recently reported approach using photoswitchable fluorescent proteins demonstrated characterization of heterogeneous multi-protein complexes at previously unthinkably low concentration ranges Chaturvedi et al 2017b). In addition, to address growing concerns from regulatory agencies regarding potential immunogenicity of therapeutic proteins, AUC-FDS can be conveniently employed to evaluate antibody-antigen interactions in close to in vivo conditions (Krayukhina et al 2017).…”