The biogenesis and maintenance of the entire endomembrane system is dependent upon membrane fusion proteins. Mounting evidence indicates that the integral membrane GTPase Atlastin is a membrane fusion protein involved in the homotypic fusion of the endoplasmic reticulum (ER) membrane suggesting a role in the biogenesis and maintenance of ER structure. I helped show that recombinant Drosophila atlastin is able to promote the fusion of synthetic membranes in vitro and that this fusion is dependent upon atlastin GTPase activity. The structure-function experiments presented here assist in elucidating domains required in the mechanism of atlastin mediated membrane fusion. ER homotypic fusion is dependent upon the self-association of Atlastin subunits in adjacent membranes to bring the bilayers into close molecular contact. Atlastin dimerization occurs in the presence of GTPγS but not GDP and stable dimerization is dependent upon a juxtamembrane middle domain three-helix bundle (3HB). The atlastin GTPase domain and 3HB form a potent soluble domain inhibitor of atlastin homotypic fusion, while the GTPase domain alone shows little inhibition. Designed GTPase domain mutations show that GTP binding and atlastin dimerization is insufficient to support fusion without GTP hydrolysis. Additionally, domain analysis of atlastin reveals that the C-terminal cytoplasmic domain of atlastin is absolutely required for membrane fusion, possibly through a protein-lipid interaction of an amphipathic alpha-helix. Genetic lesions in the human Atlastin-1 gene, SPG3A, result in a form of autosomal dominant hereditary spastic paraplegia (HSP). A better understanding of Atlastin function should lend significant insight into normal ER biogenesis and maintenance, as well as the pathology of human disease.

Many alpha-helical membrane proteins contain internal symmetries, indicating that they might have evolved through a gene duplication and fusion event Here, we have characterized internal duplications among membrane proteins of known structure and in three complete genomes We found that the majority of large transmembrane (TM) proteins contain an internal duplication The duplications found showed a large variability both in the number of TM-segments included and in their orientation Surprisingly, an approximately equal number of antiparallel duplications and parallel duplications were found However, of all 11 superfamilies with an internal duplication, only for one, the AcrB Multidrug Efflux Pump, the duplicated unit could be found in its nonduplicated form An evolutionary analysis of the AcrB homologs indicates that several independent fusions have occurred, including the fusion of the SecD and SecF proteins into the 12-TM-protein SecDF in Brucella and Staphylococcus aureus In one additional case, the Vitamin B-12 transporter-like ABC transporters, the protein had undergone an additional fusion to form protein with 20 TM-helices in several bacterial genomes Finally, homologs to all human membrane proteins were used to detect the presence of duplicated and nonduplicated proteins This confirmed that only in rare cases can homologs with different duplication status be found, although internal symmetry is frequent among these proteins One possible explanation is that it is frequent that duplication and fusion events happen simultaneously and that there is almost always a strong selective advantage for the fused form / <p>authorCount :4</p>

alpha-Helical membrane proteins are important for many biological functions. Due to physicochemical constraints, the structures of membrane proteins differ from the structure of soluble proteins. Historically, membrane protein structures were assumed to be more or less two-dimensional, consisting of long, straight, membrane-spanning parallel helices packed against each other. However, during the past decade, a number of the new membrane protein structures cast doubt on this notion. Today, it is evident that the structures of many membrane proteins are equally complex as for many soluble proteins. Here, we review this development and discuss the consequences for our understanding of membrane protein biogenesis, folding, evolution, and bioinformatics. / <p>authorCount :3</p>

Membrane proteins are involved in a number of disease pathologies and thus comprise a large number of drug targets. Determination of the high-resolution three-dimensional structure is essential for rational drug design, but several hurdles need to be overcome, primarily the over-expression and purification of said membrane proteins. Human leukotriene C4 synthase (hLTC4S), an 18 kDa integral membrane protein localized in the outer nuclear membrane of eosinophils and basophils, catalyzes the conjugation of LTA4 and reduced glutathione to produce LTC4. LTC4 and its metabolites LTD4 and LTE4 are the cysteinyl leukotrienes implicated in bronchoconstriction and inflammation pathways. The focus of my project involves optimizing the over-expression and purification of hLTC4S, which was heterologously expressed in Schizosaccharomyces pombe, purified by immobilized affinity chromatography, and finally "polished" with a buffer exchange step to remove excess co-purified lipids. The optimized protocol yielded ~1 mg of ~90% homogenous, pure protein per liter of cell culture. The finalized purified protein can then be used for further investigation of two-dimensional crystals by electron crystallography with the overall goal of structure determination.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen known to cause a variety of infections that are difficult to treat due to extremely high resistance to almost all antibiotics currently in clinical use. One of the major contributors to this resistance is the active efflux of antibiotics from the cell, primarily by action of the Resistance Nodulation Division (RND) family of efflux pumps. These pumps are composed of three proteins; an inner membrane RND pump, a periplasmic membrane fusion adaptor protein, and an outer membrane protein. The mechanism by which the three proteins interact to form a functional complex is largely unknown and the methods currently available for their study involves expression systems geared for high levels of expression. In the case of membrane proteins which play a role in clinically relevant activities, such as multidrug resistance, an expression system which does not always reflect biologically relevant levels of protein in the cell is not ideal for studying their interactions as correlation of conclusions from interaction studies to true interactions may not be possible. In this study a single copy gene expression system was designed and demonstrated to better reflect clinically relevant levels of overexpression compared to a multi-copy expression system. Quantitative-real time PCR analysis of C-terminally hexa-histidine tagged outermembrane protein, OpmH, expression shows approximately 100-fold and 20-fold overexpression from multi-copy and single-copy expression systems respectively. OpmH-H6 was successfully purified from both multi copy and single copy expression systems with proportionate purification schemes indicating the feasibility of single copy expression systems for the study of membrane bound protein complexes. / UOIT

The motivation of this work was to produce novel analytical techniques capable of probing the physical properties of the cell surface. Many researchers have used supported lipid bilayers (SLBs) as models to study the structure and function of the cell membrane. The complexity of these models is consistently increasing in order to better understand the myriad of physiologically relevant processes regulated by this surface. In order to aid researchers in studying such phenomenon, the following contributions were made. To manipulate components within the cell membrane, an electrophoretic flow cell was designed which can be used as a probe to study the effect of electrical fields on charged membrane components and for the separation of these components. This devise allows for the strict control of pH and ionic strength as species are observed in real-time using fluorescence microscopy. Additionally, advancements have been made to the production of patterned heterogeneous SLBs for use in separations and to probe the interactions of membrane components. The methodology to couple SLB separations and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) imaging was devised. This technology allows for the label-free mapping of the SLB surface post electrophoresis in order to observe naturally occurring species unperturbed by the addition of extrinsic tags. The final contribution, and perhaps the greatest, is the development of a procedure to create highly mobile SLBs from native membranes. These surfaces have vast potential in that they are no longer simple models of the cell surface, they are in fact the actual cell surface made planar. This advancement will be of great use to biophysicists and biochemists interested in using surface specific analytical methods to better understand physiological processes. These highly mobile native membrane surfaces have been coupled with the SLB electrophoresis technology to separate discrete bands of lipids and proteins, a proof of principle that will hopefully be further developed into a standard method for membrane proteomic studies. Collectively the tools and methodologies described herein show great potential in allowing researchers to further add to mankind’s understanding of the cellular membrane.

Atomic force microscopy (AFM) is a powerful technique that enables to study biological macromolecules and dynamic biological processes at different scales. It is an excellent tool for imaging of biological objects under various conditions at a nanometer resolution. Force mode of AFM, so called single molecule force spectroscopy (SMFS), allows for investigation of the strength of molecular interactions of different origins established between and within biological molecules. In the present work, SMFS was used to detect and locate structurally and functionally important interactions of sodium/glycine betaine transporter BetP of Corynebacterium glutamicum, which serves as a model system for this class of proteins. Mechanical pulling of BetP molecules embedded into the lipid membranes resulted in a step-wise unfolding of the protein and revealed insights into its structural stability. Effect of the lipid environment, N- and C-terminal extensions on inramolecular interactions of BetP as well as protein activation and ligand binding were investigated in great detail. In another part of this work, I demonstrate an application of the AFM based technique that can record unfolding of a protein under force-clamp conditions. This method directly measures the kinetics of the protein unfolding, allowing for the use of simple methods to analyze the data. For the first time the force-clamp technique was used to describe in detail unfolding kinetics of the membrane protein, i. e. Na+/H+-antiporter NhaA from Escherichia coli. Performed here experiments on NhaA in its functionally active and inactive states demonstrated the advantages of examining unfolding kinetics at the single-molecule level. It was possible to observe unfolding events for pH-activated conformation of NhaA that due to the low frequency of occurrence were not represented in the ensemble average of the single-molecule measurements. As mechanical unfolding, similarly to bond rupture, is a force-dependent process, force-clamp technique can allow for a more direct way of probing protein unfolding and is anticipated to be also useful to examine the folding/unfolding kinetics of other membrane proteins.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "August 2001." Typescript. Vita. Includes bibliographical references (leaves 135-149). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 100-114).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 184-198).