<p>Cell membranes are dynamic structures with complex organization. The complexity of the cell membrane arises from intrinsic membrane structure, membrane microdomains within the plasma membrane and the membrane cytoskeleton. Plasma membrane receptors are integral membrane proteins with diverse structures and functions which bind specific ligands to trigger cellular responses. Due to compartmentalization of the plasma membrane and the formation of membrane microdomains, receptors are distributed non-homogeneously in the cell membrane bilayer. Both lateral and rotational diffusion of membrane receptors reflects different kinds of intermolecular interactions within the plasma membrane environment. Understanding protein diffusion within the membrane is very important to further understanding biomolecular interactions in vivo during complex biological processes including receptor-mediated signaling. </p><p> Rotational diffusion depends linearly on the in-membrane volume of the rotating proteins. Relative to lateral diffusion, rotational diffusion is a more sensitive probe of an individual molecule&rsquo;s size and local environment. We have used asymmetric quantum dots (QD) to conduct imaging measurements of individual 2H3 cell Type I Fc&epsiv; receptor rotation on timescales down to 10 msec per frame. We have also used time-tagged single photon counting measurements of individual QD to examine &micro;sec timescales, although rapid timescales are limited by QD emission rates. In both approaches, decays of time-autocorrelation functions (TACF) for fluorescence polarization fluctuations extend into the millisecond timescale, as implied by time-resolved phosphorescence anisotropy results. Depending on instrumental parameters used in data analysis, polarization fluctuation TACFs can contain a contribution from the intensity fluctuation TACF arising from QD blinking. Such QD blinking feed-through is extremely sensitive to these analysis parameters which effectively change slightly from one measurement to another. We discuss approaches based on the necessary statistical independence of polarization and intensity fluctuations to guarantee removal of a blinking-based component from rotation measurements. Imaging results demonstrate a range of rotational behavior among individual molecules. Such slow motions, not observable previously, may occur with large signaling complexes, which are important targets of study in cell biology. These slow motions appear to be a property of the membrane itself, not of the receptor state. Our results may indicate that individual mesoscale membrane regions rotate or librate with respect to the overall cell surface. </p><p> The luteinizing hormone receptor (LHR) is a seven transmembrane domain receptor and a member of the GPCR family. It is located on luteal cells, granulosa and theca cells in females. Understanding how these protein receptors function on the plasma membrane will lead to better understanding of mammalian reproduction. LHR becomes aggregated upon binding hCG when receptors are expressed at physiological numbers. Binding of hormone to LHR leads to activation of adenylate cyclase (AC) and an increase in intracellular cyclic AMP (cAMP). ICUE3 is an Epac-based cAMP sensor with two fluorophores, cyan fluorescent protein (CFP) and the YFP variant, cpVenus, and a membrane-targeting motif which can be palmitoylated. Upon binding cAMP, ICUE3 undergoes a conformational change that separates CFP and YFP, significantly reducing FRET and thus increasing the ratio of CFP to YFP fluorescence upon excitation with an arc lamp or 405nm laser source. Hence we have investigated hLHR signal transduction using the cyclic AMP reporter probe, ICUE3. A dual wavelength emission ratio (CFP/YFP) imaging method was used to detect a conformational change in ICUE3 upon binding cAMP. This technique is useful in understanding the sequence of intercellular events following hormone binding to receptor and in particular, the time course involved in signal transduction in a single cell. Our data suggested that CHO cells expressing ICUE3 and directly treated with different concentrations of cAMP with saponin can provide a dose-dependent relationship for changes in intracellular cAMP levels. Forskolin (50&micro;M) causes maximal activation of the intracellular cAMP and an increase in the CFP/YFP emission ratio. In CHO cells expressing both ICUE3 and hLHR-mCherry, the CFP/YFP ratio increased in cells treated with forskolin and in hCG- treated cells. In flow cytometry studies, similar results were obtained when CHO cells expressed &lt; 60k LHR-mCherry per cell. Our results indicate that ICUE3 can provide real time information on intracellular cAMP levels, and the ICUE3 is a reliable cAMP reporter can be used to examine various aspects of LH receptor-mediated signaling. </p>

Thesis (Ph.D.)--University of California, San Francisco, 2005. / Includes bibliographical references. Also available online.

Thesis (PH.D.) -- Syracuse University, 2004. / "Publication number AAT 3135882."

Thesis (PH.D.) -- Syracuse University, 2006 / "Publication number AAT 3241860."

<p> Hsp90 is a highly conserved, ATP-dependent molecular chaperone that is essential for maintaining the functions of its client proteins. It has been estimated that about 10% of the proteome of a eukaryotic cell interacts with Hsp90. A large subset of this portion consists of protein kinases and steroid hormone receptors, putting Hsp90 as the master regulator of many essential cellular functions. The mechanism of how Hsp90 uses ATP hydrolysis to carry out its function remains unclear. Structural studies of Hsp90 revealed that Hsp90 is a V-shaped homodimer with each protomer composed of three well-folded domains: an ATP-binding N-terminal Domain (NTD), a Middle Domain (MD), and a C-terminal dimerization Domain (CTD). Efficient ATP hydrolysis by Hsp90 requires that the dimer forms a closed state where NTDs are dimerized, forming a closed &rdquo;clamp&rdquo; conformation that is stabilized by ATP binding. The kinetics of forming this stably closed state is not driven by ATP binding, as there are other rate-limiting steps that need to occur within the protein to allow the NTDs to be dimerized. So how does Hsp90 actually use the energy from ATP to remodel its client protein? the focus of this thesis examines the other possibility of this happening during ATP hydrolysis. In the first chapter, I followed up an observation made by a previous graduate student Laura Lavery. She observed that the ATP-bound closed state of a mitochondrial Hsp90 (TRAP1) is asymmetric. The asymmetry is most prominent at the juncture between the MD:CTD interface&mdash;one protomer is buckled while the other remains straight, resembling the same conformation previously observed in the symmetric closed &rdquo;clamp&rdquo; state. This buckling happens precisely where conserved binding sites have been mapped for client proteins. This suggests that if conformational changes were to occur due to ATP hydrolysis, subsequent rearrangements of the asymmetric MD:CTD interfaces back to the previously observed symmetric closed state vii could be used to drive client protein remodeling. Using a combination of biophysical methods (crystallography, Double Electron-Electron Resonance (DEER), and FRET), we observed that TRAP1 hydrolyzes the 2 bound ATPs sequentially. The buckled protomer hydrolyzes the first ATP, which is then followed by a flip in the asymmetry (the buckled conformation becomes straight and vice versa, on each side), which primes the second ATP for hydrolysis by a buckled protomer. In this model, the MD:CTD interface is guaranteed to undergo remodeling with each ATP hydrolysis and would make efficient use of energy from ATP. The implications for this asymmetric ATP hydrolysis mechanism may also be relevant to other Hsp90s. While we have not observed any other asymmetric Hsp90 structures by itself, several functional Hsp90 complexes seen so far seem to have asymmetric composition/arrangements of their components. In the second chapter, we explore how TRAP1 ATPase activity can be modulated by different divalent cations as co-factors. Despite having two ATP binding sites, the ATPase activity of most Hsp90 homologs appears to be non-cooperative (each site behaves independently from one another). However, we saw that ATPase activity of TRAP1 can be cooperative in presence of calcium, and the activity in presence of magnesium appears to be bi-phasic, with higher activity at low ATP concentrations. This unique behavior of TRAP1 may yet be another adaptation of the Hsp90 machine that has evolved within the mitochondrial matrix environment. Using crystallography, we also discovered that calcium binds to the NTD of TRAP1 unlike previously observed chaperone/calcium/ATP complexes. While the exact biological role for this phenomena is not yet clear, these findings provide a clear molecular basis for the regulation of TRAP1 by calcium. Taken together, the work described in this thesis provide insights into the mechanism of ATP hydrolysis by Hsp90, and a potential role that TRAP1 plays in calcium/magnesium-regulated mitochondrial physiology.</p><p>

<p> Apolipoprotein E is an exchangeable apolipoprotein whose isoforms are associated with various disease risk profiles. Individuals bearing the <i> APOE</i> &epsiv;4 allele are at increased risk for developing Alzheimer&rsquo;s disease compared to those bearing the <i>APOE</i> &epsiv;3 allele. The two isoforms differ in amino acid at position 112: apoE3 bears a Cys while apoE4 bears an Arg. It is hypothesized that the Cys to Arg substitution in apoE4 causes a decrease in stability in comparison to apoE3, which is exaggerated at endosomal pH &lt;6.0. In our study, changes in secondary structure were monitored using circular dichroism at pH 7.4 and pH 3.5. Chemical denaturation indicated that both apoE3 and apoE4 retained their helical secondary structure at the lower pH, with a biphasic and monophasic guanidine HCl denaturation profile, respectively. Tertiary structure was monitored at both pH&rsquo;s through fluorescence spectral characteristics and mobility of a fluorescent probe attached to each of the 7 major amphipathic &alpha;-helices of apoE3 and apoE4. The data showed decreases in fluorescence emission (FE), changes in fluorescence polarization (FP), and fluctuations in probe mobility, which were interpreted as likely formation of a molten globule. Formation of a molten globule appeared to occur during denaturation primarily for apoE4, and thermodynamic parameters of apoE4 showed a lower stability than apoE3, with a larger effect of pH. Taken together, our results suggest that the acidic pH in the endosomal compartments could interact with the native structure of apoE4 to generate a molten globule state that is able to bind endosomal membranes, other proteins, or itself. This study offers mechanistic insight into the impact of the single residue difference between apoE3 and apoE4 with regard to folding/unfolding behavior, and with regard to its physiological and pathological implications.</p><p>

<p>A vast majority of cell signaling is mediated through activation of hetero-trimeric G proteins. G-Interacting Vesicle associated protein (GIV) is a non-receptor Guanine nucleotide exchange factor (GEF), which activates Gi family of heterotrimeric G proteins downstream of activated receptor tyrosine kinases (RTKs). GIV?s GEF function is mediated by a stretch of highly conserved ~20 residues, which is followed by a putative SH2-like domain in the C-terminus of the protein. Previous studies have shown that the C-terminal 211 amino acids of GIV (referred to as ?GIV-CT? henceforth) are capable of functioning autonomously from its recruitment to the cell surface upon activation of RTKs promoting downstream signals by binding to and activating Gi proteins. However, despite all the functional information and computational predictions, the structural insights into how GIV-CT is able to perform all these functions is missing. Here, we have attempted to probe the structural aspects of GIV-CT using circular dichroism (CD) spectroscopy ? the most commonly used method for determining the secondary structure of peptides and proteins. N-terminally His6-tagged GIV-CT wild type (WT) and a phosphomimetic mutant (S1674D; binds and activates Gi better than the WT) were expressed and purified from E. coli BL21-DE3 cells using Co2+-NTA affinity chromatography followed by cation exchange chromatography. Our preliminary CD spectroscopy analyses showed a random coil profile for GIV-CT WT as well as S1674D mutant, both of which could be induced to adopt an -helical conformation by addition of trifluoroethanol (TFE). Further analysis of CD spectra to predict secondary structure characteristics was carried out using DichroWeb, an online deconvolution program, which compares CD data for known protein structure to that of unknown protein structure. Wild type GIV-CT secondary structure was predicted to contain 1% ?-helix, 19% ?-sheet and 47% random coil while our mutant contained 1% ?-helix, 19% ?-sheet and 43% random coil. -turn and `denatured? helix and sheet percentages make up the final total of 100% secondary structure. In the presence of TFE, GIV-CT WT and S1674D ?-helical content increased to 23% and 25%, respectively. Together, our data suggest that although recombinant GIV-CT may be predominantly in an unstructured state, it likely has a propensity to fold into a regular secondary structure, perhaps upon interacting with a binding partner.

The Hsp70 family molecular chaperones prevent protein aggregation under heat shock conditions. They are highly conserved, and have a N-terminal ATPase domain, which binds and hydrolyzes ATP, and a C-terminal substrate-binding domain, which binds unfolded stretches of polypeptides. The communication between the two domains is vital for chaperone function. The nucleotide state regulates the substrate affinity, and substrate binding stimulates the ATP hydrolysis rate. Our study to understand this allosteric mechanism showed that the interdomain linker region is crucial for the stimulation of the ATPase activity. The linker region between the two domains is highly conserved and hydrophobic, and when retained with the ATPase domain (DnaK1–392), poised the domain in the high ATPase activity state (8-fold higher activity). The ATPase domain without the linker residues (DnaK1–388) has ATPase activity comparable to the basal ATPase activity of the intact wild-type protein. When these linker residues are mutated in the ATPase domain construct (DnaK1–392(L390D/L391D)), the stimulation of the ATPase activity decreases (2-fold higher activity). The same linker mutation in intact DnaK (DnaK1-638(L390D/L391D)) disrupts the interdomain relationship and the propagation of the allosteric communication in both directions. Therefore, the linker region is the allosteric switch that mediates the communication between the ATPase domain and the substrate-binding domain. In addition to the linker region, there are other structural elements, which are part of the interdomain interface, and we tried to identify these regions by fluorescence experiments. In the ATP-bound state, the two domains are more intimately associated, and the α-helical subdomain of the substrate-binding domain and subdomains IA and IB of the ATPase domain are forming interdomain contacts. In the ADP-bound state, the N-terminus of the bound substrate peptide is within 30 Å of W102 in the ATPase domain. Several mutations in the substrate-binding domain destabilize the substrate-binding domain and push the conformational equilibrium towards the docked ATP-bound state. This showed that the Hsp70 proteins maintain a delicate equilibrium in order to function. Based on our data, we have proposed a structural model for the allosteric mechanism of DnaK.

Transport of proteins across membranes is essential during many stages of pathogen infection and colonization of human cells. Many Gram-negative pathogens use a Type 3 Secretion (T3S) system to inject proteins into the target cell during infection. Substantial genetic and biochemical evidence suggest that proteins are translocated across the host plasma membrane through a proteinaceous pore or translocon formed by two bacterial secreted proteins: the T3S translocators. Despite its key role in pathogenesis, virtually nothing is known about the assembly mechanism, structure, and composition of this critical transmembrane complex. To this end, a cell-free system for the structural and functional characterization of Pseudomonas aeruginosa T3S translocators PopB and PopD was established. PopB and PopD assemble discrete sized pores in liposomal membranes. These pores are stable and heteromeric in nature. Combining this reconstitution methodology with single-molecule fluorescence microscopy methods, the stoichiometry of the membrane-assembled hetero-complex was determined: PopB and PopD assemble an hexadecameric complex at the membrane, with a calculated molecular weight of 601 kDa. The obtained stoichiometry is consistent with ex vivo estimations of the translocon size, and represents the first report on the stoichiometric arrangement of a Type 3 Secretion System translocon.

Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's Diseases. Caspase-6 is of great interest in treatment of these neurodegenerative diseases, however the structure and molecular events of caspase-6 activation remained poorly understood prior to this thesis work. To study the activation events and to probe the role of the prodomain and intersubunit linker on caspase-6 structure and activation, we generated a series of caspase-6 cleavage variants. Autoprocessing at D193 of the intersubunit linker proved to be crucial for activity and for subsequent processing at D179 site. For the first time we report the existence of stabilizing interactions between the prodomain and intersubunit linker of caspase-6. We reported one of the first crystal structures of ligand-free caspase-6 with and without the intersubunit linker. Mature caspase-6 has extended 60.s and 130.s regions and the 90.s helix is rotated outwards. These striking structural features are unique to caspase-6 and are not observed in any other caspase. Caspase-6 shows a loss of helicity upon binding substrate. Therefore, binding of substrate appears to cause a conformational change, potentially converting the 60.s and 130.s helices to a canonical caspase structure. Recent crystal structure of active-site bound caspase-6, underscore our findings about the requirement of conformational changes to bind substrate. Our work suggests that the latent state of mature caspase-6 relies on the stability and helical propensity of the 130.s region. The presence of the helical sequence in caspase-6 is more critical for formation of the extended conformation than is the helixbridging network between the 60.s and 130.s helices. This extended 130.s helix in the mature caspase-6 causes it to transition through a less stable intermediate to bind substrate. All these unique structural features reflect on the enigmatic role of caspase-6 in the cell. The conformational changes between the apo mature and bound states are likely to be important in designing caspase-6 selective therapies. Thus our study has enhanced the current knowledge about caspase-6 structure and function and broadened our understanding of caspase activation and regulation in general.