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Membrane protein crystallization in the lipid cubic phase: testing hypotheses relating to reconsitution

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2007, Doctor of Philosophy, Ohio State University, Biophysics.
The in meso method, also called as lipidic cubic phase, has been proved to be a useful technique for membrane protein crystallization (Caffrey, 2003). The method was introduced in 1996 by using bacteriorhodopsin (bR) as the target membrane protein (Landau and Rosenbusch, 1996). More and more membrane proteins’ structures have been solved by the in meso method thereafter, including halorhodopsin (Kolbe et al., 2000), sensory rhodopsin (Luecke et al., 2001), the sensory rhodopsin/transducer complex (Gordeliy et al., 2002), the photosynthetic reaction center from Rhodobacter sphaeroides (Katona et al., 2003), the cobalamin transporter (BtuB) (Cherezov et al., 2006), the light harvesting 2 complex (Cherezov et al., 2006), the outer membrane adhesion protein (OpcA) (Cherezov et al., 2007a), and more recently one GPCR protein (Cherezov et al., 2007b). While the in meso method becomes more productive in membrane protein structural studies, its mechanism remains unknown. However, the working hypothesis has been advanced in 2000 (Caffrey, 2000), in which suggests that the membrane protein will reconstitute in the lipid bilayer prior to the final nucleation and crystallization process. In order to investigate this step, fluorescence quenching technique was brought into lipidic cubic phase sample. Hydrophobic peptides were employed as the target molecules for the initial studies. After successfully certified the reconstitution of the hydrophobic peptide into lipid bilayer in the cubic phase, the studies were extended into membrane protein molecules. bR, BtuB and OpcA were chosen for this purpose, and all spectroscopic results support the reconstitution process. In order to better understand the reconstitution process, the transport properties of the lipidic cubic phase were studies by using tryptophan as the target molecule. And the residue has been alkylated in the structure and repeated in the sequence to enlarge the interaction between lipid bilayer and target molecule. Partition and release data were collected for this purpose. Another hypothesis regarding to the cubic phase sample is to refold the inclusion bodies (IB) of membrane protein in it and use it for the crystallization trials. Some initial studies to examine this hypothesis were introduced at the end of this dissertation.
Ross Dalbey (Advisor)

Recommended Citations

Citations

  • Liu, W. (2007). Membrane protein crystallization in the lipid cubic phase: testing hypotheses relating to reconsitution [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1196274127

    APA Style (7th edition)

  • Liu, Wei. Membrane protein crystallization in the lipid cubic phase: testing hypotheses relating to reconsitution. 2007. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1196274127.

    MLA Style (8th edition)

  • Liu, Wei. "Membrane protein crystallization in the lipid cubic phase: testing hypotheses relating to reconsitution." Doctoral dissertation, Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1196274127

    Chicago Manual of Style (17th edition)