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Rearrangements of The First Transmembrane Helix of The Plasma Membrane H-ATPase
Bassem A. Gayed
Biomedical Engineering Program
B.S. 2005, New Jersey Institute of Technology
Thesis Advisor: Joshua R. Berlin, Ph.D.
Department of Pharmacology & Physiology
Thursday, October 27, 2011
6:00 P. M., MSB H609b
P-type ATPases are a large family of integral membrane proteins that maintain the electrochemical gradient across cellular membranes via active transport. Since the discovery of the first member of this family (Na,K-ATPase) by Jens Skou in 1957, our understanding of the mechanism involved in their enzymatic reaction has greatly increased. The recent availability of crystal structures of several members of the P-type ATPases combined with the wealth of biochemical data has allowed for a detailed proposal of the enzymatic reaction taking place during the transport process. However, the conservation of the reaction mechanism among different members of the family is still unclear.
We focused in this study on the similarities between two members of the P-type ATPases that have different ion specificity. We used the SERCA Ca-ATPase, which has been the most studied member of the family to predict the conformational changes that the plasma membrane H-ATPase undergoes during its reaction cycle. We provided evidence that conformational changes taking place in the transmembrane region of the H-ATPase mimic the proposed conformational changes of SERCA. We genetically incorporated the environmentally-sensitive fluorescent unnatural amino acid, prodan AA, into specific locations in the Arabidopsis thaliana type 2 H-ATPase (AHA2) expressed in Saccharomyces cerevisiae. We showed that we can introduce the fluorescent unnatural amino acid into the transmembrane region of the protein and, by taking advantage of the environmentally sensitive properties of prodan AA, the resultant labeled protein provided valuable information about the local environment surrounding the incorporated fluorescent probe.
Furthermore, we monitored the change of accessibility of cysteines introduced into the first transmembrane helix (TM1) of AHA2 to the extracellular environment by using thiol-reactive probes such as Fluorescein -5- Maleimide, MTSET and MTSES. We showed that the accessibility of TM1 changes during the reaction cycle of AHA2. This result is in agreement with the proposed reaction scheme of SERCA. This work increased our understanding of the transport mechanism utilized by the P-type ATPases and shed light on the similarities that might exist between various members of the family with different substrate specificity.