483 Functional Reconstitution of the Integral Membrane Enzyme, His-Ste14p, in Synthetic Bolalipid Membrane Vesicles

Friday, 23 October 2009: 2:25 PM
103-B (Puerto Rico Convention Center)
Wilma Febo-Ayala , Department of Chemistry, University of Puerto Rico-Mayaguez, Mayaguez, PR
Shakira L. Morera-Felix , Department of Chemistry, Purdue University, West Lafayette, IN
Christine A. Hrycyna , Department of Chemistry, Purdue University, West Lafayette, IN
David H. Thompson , Department of Chemistry, Purdue University, West Lafayette, IN
Studies were undertaken to understand the effect of changes in chemical structure of bolalipids on the physical properties of their membranes and the functional reconstitution of isoprenylcysteine carboxyl methyltransferase.  Three different structures of bolalipids were synthesized and studied: C20BAS, C32BAS and C32-phyt.  Differential Scanning Calorimetry and Fluorescence Recovery after Photobleaching experiments were performed on the bolalipid membranes to obtain gel to liquid-crystalline phase transition temperatures and lateral diffusion coefficients, respectively.  In vitro vapor diffusion methyltransferase assays investigate His-Ste14p transmembrane enzyme activity when reconstituted in membranes composed of C20BAS:E. Coli polar lipid, C20BAS:POPC, C32-phyt:E. Coli polar lipid, C32-phyt:POPC.  Results show that transmembrane enzyme activity is retained or even enhanced with increasing bipolar lipid content, except in membranes composed of 100% C20BAS where His-Ste14p activity was significantly reduced.  Confocal immunofluorescence microscopy confirmed the presence of the enzyme in the C20BAS membrane.  It was found that the T of the assay or phase (gel or liquid-crystal) of the lipid does not affect His-Ste14p activity.  Thus, the low enzymatic activity in C20BAS membranes is attributed to the hydrophobic mismatch between the lipid and the hydrophobic section of the enzyme.  To our knowledge, this is the first functional reconstitution of a transmembrane enzyme in synthetic bolalipids.