Title

Single Molecule Spectroscopy Studies of Membrane Protein Dynamics and Energetics by Combined Experimental and Computational Analyses

Date of Award

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Photochemical Sciences

First Advisor

Peter Lu, PhD

Second Advisor

Arthur Samel, PhD (Committee Member)

Third Advisor

John Cable, PhD (Committee Member)

Fourth Advisor

Alexander Tarnovsky, PhD (Committee Member)

Abstract

Making and holding an artificial lipid bilayer horizontally in aqueous solution within the microscopic working distance are essential for simultaneous single molecule imaging and single ion-channel electrical current recording of membrane ion-channel proteins. We have designed and developed an unsupported, horizontally held artificial bilayer and used it successfully in studying the ion-channel protein dynamics by simultaneous single-molecule fluorescence imaging and electric current measurements. Further, using the developed lipid bilayer, we have studied the behavior of colicin Ia ion channel protein. The dynamics of the channel transition between open and close states and the translocation of charged ¿¿¿¿-helices across the membrane were studied. Our results have demonstrated that the channel open-close transition is less dependent on the applied voltage and the charged 2-5 helices follow a polar pathway to cross the lipid bilayer. Moreover, we studied about the structural arrangement of light harvesting complex II (LH2) proteins in the membrane. Studies have revealed the inhomogeneous structural organization of the LH2 complexes giving evidences for the existence of energetically coupled linear LH2 aggregates in the native photosynthesis membrane of purple bacteria. Our AFM imaging results reveal the formation of linear aggregates where each monomer is tilted along the aggregated structure in photosynthetic membrane. The spectroscopic results support our attribution and the model calculation for the absorption, emission and lifetime are consistent with the experimentally determined spectroscopic values, further proving a molecular-level understanding of the LH2 structural arrangement in the photosynthetic membrane.