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0 Q&A 1849 Views Dec 20, 2021

The functional performance of a cell depends on how macromolecules, in particular proteins, come together in a precise orientation, how they assemble into protein complexes and interact with each other. In order to study protein-protein interactions at a molecular level, a variety of methods to investigate these binding processes yield affinity constants and/or the identification of binding regions. There are several well-established biophysical techniques for biomolecular interaction studies, such as fluorescence spectroscopy and surface plasmon resonance. Although these techniques have been proven to be efficient, they either need labeling or immobilization of one interaction partner. Backscattering interferometry (BSI) is a label-free detection method, which allows label- and immobilization-free interaction analysis under physiologically relevant conditions with high sensitivity and in small volumes. We used BSI to measure the interaction of the neuronal calcium sensor recoverin with its target G protein-coupled receptor kinase 1 (GRK1) as a model system. Increasing concentrations of purified recoverin were mixed with a specific concentration of a GRK1 fusion protein. In this protocol, we provide a full description of the instrumental setup, data acquisition, and evaluation. Equilibrium dissociation constants of recoverin-GRK1 interaction determined by the BSI instrumental setup are in full agreement with affinity constants obtained by different methods as described in the literature.

0 Q&A 2132 Views Oct 20, 2021

Diffusion is a fundamental process in biological systems that governs the molecular collisions driving biochemical reactions and membrane and transport. Measurement of the diffusion coefficient and application of the Stokes-Einstein equation produces the hydrodynamic radius, which is a commonly used gauge of particle size. Additionally, measurement of the diffusion coefficient and the sedimentation coefficient, and application of the Svedberg equation, yields the molecular weight, which is particularly useful in the characterization of very large macromolecules. Dynamic light scattering (DLS) is the most common method to measure the diffusion coefficient of macromolecules. We describe a procedure to perform DLS measurements on monomeric bovine serum albumin (BSA) purified by size-exclusion chromatography using the Zetasizer Nano S particle size analyzer. We compare several analytical methods in existing software programs to estimate the diffusion coefficient of BSA (extrapolated to water at 20°C at infinite dilution, ) and describe a statistical method to obtain 95% confidence limits of the precision of the estimates. We compare estimates to literature values obtained by diffusiometry, sedimentation velocity analytical ultracentrifugation, and other DLS instruments. The method of cumulant analysis in the program SEDFIT ( produced the most precise estimate, 6.06 ± 0.07 F (1 F = 10-7 cm2 s-1), which was within the range of estimates obtained by diffusiometry or sedimentation velocity. This protocol is useful for DLS method validation and quality control.

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