Abstract
Membrane proteins mediate a number of cellular functions and are associated with several diseases and also play a crucial role in pathogenicity. Due to their importance in cellular structure and function, they are important drug targets for ~60% of drugs available in the market. Despite the technological advancement and recent successful outcomes in determining the high-resolution structural snapshot of membrane proteins, the mechanistic details underlining the complex functionalities of membrane proteins is least understood. This is largely due to lack of structural dynamics information pertaining to different functional states of membrane proteins in a membrane environment. Fluorescence spectroscopy is a widely used technique in the analysis of functionally-relevant structure and dynamics of membrane protein. This review is focused on various site-directed fluorescence (SDFL) approaches and their applications to explore structural information, conformational changes, hydration dynamics, and lipid-protein interactions of important classes of membrane proteins that include the pore-forming peptides/proteins, ion channels/transporters and G-protein coupled receptors.
Highlights
Biological membranes are composed of heterogeneous mixtures of lipids and proteins which facilitate cellular compartmentalization for specialized functions
The environment sensitivity of tryptophan has been shown to be useful in defining the ends of transmembrane helices as in the case of mechanosensitive channel of large conductance, MscL (Powl et al, 2005). These studies clearly highlight the potential of such site-directed fluorescence approaches in dynamic structural biology of membrane proteins
The advantage of this method is that it can provide the depth of the protein or peptide in angstrom resolution. This quenching approach has been widely used to characterize the membrane penetration depths of intrinsic Trp residues and other extrinsic probes labeled at specific sites in nicotinic acetylcholine receptor (Chattopadhyay and McNamee, 1991), hemolytic peptide melittin (Ghosh et al, 1997; Raghuraman and Chattopadhyay, 2004c, 2007b; Haldar et al, 2008), cholesterol oxidase (Chen et al, 2000), ricin (Ramalingam et al, 1994), calcium dependent membrane binding protein annexins (Meers, 1990), model ion channel peptide (Chung et al, 1992) and colicin (Palmer and Merrill, 1994)
Summary
Biological membranes are composed of heterogeneous mixtures of lipids and proteins which facilitate cellular compartmentalization for specialized functions. Like Trp and NBD, bimane is a small (comparable to the size of Trp), environment-sensitive probe (Figure 3) with well-characterized spectral properties (Kosower et al, 1982; Mansoor et al, 2010) and has been widely used to monitor the functionally-relevant dynamic structural changes in several membrane proteins (Islas and Zagotta, 2006; Yao et al, 2006; Semenova et al, 2009; Tsukamoto et al, 2009; Tsukamoto and Farrens, 2013). The environment sensitivity of tryptophan has been shown to be useful in defining the ends of transmembrane helices as in the case of mechanosensitive channel of large conductance, MscL (Powl et al, 2005) These studies clearly highlight the potential of such site-directed fluorescence approaches in dynamic structural biology of membrane proteins. A typical β-hairpin will show alternating aqueous and nonaqueous environment variation and the quenching pattern can be subjected to helical wheel analysis to obtain the helical nature of the peptide or protein (Heuck and Johnson, 2002)
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