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How to quantify protein diffusion in the bacterial membrane
Author(s) -
van den Wildenberg Siet M. J. L.,
Bollen Yves J. M.,
Peterman Erwin J. G.
Publication year - 2011
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.21585
Subject(s) - fluorescence recovery after photobleaching , fluorescence correlation spectroscopy , membrane , chemistry , diffusion , biophysics , membrane protein , biological system , bacteria , photobleaching , chemical physics , fluorescence , biochemistry , molecule , biology , physics , genetics , organic chemistry , quantum mechanics , thermodynamics
Lateral diffusion of proteins in the plane of a biological membrane is important for many vital processes, including energy conversion, signaling, chemotaxis, cell division, protein insertion, and secretion. In bacteria, all these functions are located in a single membrane. Therefore, quantitative measurements of protein diffusion in bacterial membranes can provide insight into many important processes. Diffusion of membrane proteins in eukaryotes has been studied in detail using various experimental techniques, including fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and particle tracking using single‐molecule fluorescence (SMF) microscopy. In case of bacteria, such experiments are intrinsically difficult due to the small size of the cells. Here, we review these experimental approaches to quantify diffusion in general and their strengths and weaknesses when applied to bacteria. In addition, we propose a method to extract multiple diffusion coefficients from trajectories obtained from SMF data, using cumulative probability distributions (CPDs). We demonstrate the power of this approach by quantifying the heterogeneous diffusion of the bacterial membrane protein TatA, which forms a pore for the translocation of folded proteins. Using computer simulations, we study the effect of cell dimensions and membrane curvature on measured CPDs. We find that at least two mobile populations with distinct diffusion coefficients (of 7 and 169 nm 2 ms −1 , respectively) are necessary to explain the experimental data. The approach described here should be widely applicable for the quantification of membrane‐protein diffusion in living bacteria. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 312–321, 2011.