Mechanistic pathway

Examine Membrane Protein Translocation Pathway UsingFluorescence

Mechanistic pathways by which the membrane bound or embedded protein achieves channel formation are one of the most fundamental questions asked in cell biology. Protein conducting channel at the membrane is one of the most studied of systems, however, such processes are the least understood due to complex nature and the mechanism involved. Structural information about membrane bound protein provides insight into many important aspects of formation, and it is important in order to understand the biophysical and biochemical aspects of protein conformation. In order to achieve the goal of accurately investigate the structure, functional roles and regulatory of a membrane bound orembeddedprotein. One must use multiple independent fluorescence techniques to characterize structures of a functional protein under its native conditions which cannot be acquired by X-ray crystallography and NMR technique [1]. Nevertheless, fluorescence mapping also provide comprehensive information from kinetic to thermodynamic, about the assembly mechanism, function and structure, among others.

Recently, several important structure questions about mitochondrial TIM23 complex were addressed using independent fluorescence mapping technique. As of which individual transmembrane domains form the protein conducting channel? Do the helices face an aqueous channel or a lumen environment and does the presence of substrate affect the conformational and change the environment of the helices [2, 3]. Using methodology mentioned previously [4-6] to incorporate a 7-nitrobenz-2-oxa-1,3-diazolyl (NBD) fluorescent dye into a specific Cystein site in Tim23 by NBD labeled aminoacyl-tRNA, NBD-[14C]Cys-tRNAcys to an in vitro translation reaction programmed with mRNA encoding a Tim23 monocystein mutant library [2].

NBD is a small uncharged fluorescent dye which displays a higher fluorescence lifetime, and a higher steady-state emission intensity when it is in a polar environment [2]. Because of its sensitivity to a specific residue's environment, one may take advantage of this fluorescent probe and ask several interesting questions regarding to the translocation process of membrane proteins. First, how does certain membrane protein incorporated into the lipid bilayer, and does it depend on or interact with other protein in order for the membrane insertion. By measuring fluorescence resonance energy transfer (FRET) between the donor from one helix and the acceptor from the other helix, the intramolecular distance can be modeled between the helices from two different proteins or within one protein. Nevertheless, a library of functional mutant proteins with differently positioned donor and acceptor pairs has to be constructed. Second, how does different environment influence the membrane protein conformation such as secondary and tertiary structure? The secondary structure state upon lipid bilayer insertion and solubilized in detergent can be examined using circular dichroism (CD). The protein has to be purified and be able to incorporate intoartificial lipid bilayers. Thisproposalmight be general interest for revealingthe translocation mechanismand the conformation changes with membrane incorporated protein.

1. Johnson, A.E., Fluorescence approaches for determining protein conformations, interactions and mechanisms at membranes. Traffic, 2005. 6(12): p. 1078-92.

2. Alder, N.N., R.E. Jensen, and A.E. Johnson, Fluorescence mapping of mitochondrial TIM23 complex reveals a water-facing, substrate-interacting helix surface. Cell, 2008. 134(3): p. 439-50.

3. Alder, N.N., et al., Quaternary structure of the mitochondrial TIM23 complex reveals dynamic association between Tim23p and other subunits. Mol Biol Cell, 2008. 19(1): p. 159-70.

4. Johnson, A.E., et al., Nepsilon-acetyllysine transfer ribonucleic acid: a biologically active analogue of aminoacyl transfer ribonucleic acids. Biochemistry, 1976. 15(3): p. 569-75.

5. Crowley, K.S., G.D. Reinhart, and A.E. Johnson, The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell, 1993. 73(6): p. 1101-15.

6. Woolhead, C.A., P.J. McCormick, and A.E. Johnson, Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell, 2004. 116(5): p. 725-36.

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