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Diffusion-based determination of protein homodimerization on reconstituted membrane surfaces

BMB Reports 2021년 54권 3호 p.157 ~ 163
Jepson Tyler A., Chung Jean K.,
소속 상세정보
 ( Jepson Tyler A. ) - Colorado State University Department of Chemistry
 ( Chung Jean K. ) - Colorado State University Department of Chemistry

Abstract


The transient interactions between cellular components, particularly on membrane surfaces, are critical in the proper function of many biochemical reactions. For example, many signaling pathways involve dimerization, oligomerization, or other types of clustering of signaling proteins as a key step in the signaling cascade. However, it is often experimentally challenging to directly observe and characterize the molecular mechanisms such interactions-the greatest difficulty lies in the fact that living cells have an unknown number of background processes that may or may not participate in the molecular process of interest, and as a consequence, it is usually impossible to definitively correlate an observation to a well-defined cellular mechanism. One of the experimental methods that can quantitatively capture these interactions is through membrane reconstitution, whereby a lipid bilayer is fabricated to mimic the membrane environment, and the biological components of interest are systematically introduced, without unknown background processes. This configuration allows the extensive use of fluorescence techniques, particularly fluorescence fluctuation spectroscopy and single-molecule fluorescence microscopy. In this review, we describe how the equilibrium diffusion of two proteins, K-Ras4B and the PH domain of Bruton's tyrosine kinase (Btk), on fluid lipid membranes can be used to determine the kinetics of homodimerization reactions.

키워드

Dimerization; Fluorescence correlation spectroscopy; In vitro membrane reconstitution; Single molecule tracking

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