Computational Studies of Protein Structure, Dynamics, and Function in Native-like Environments

Proteins are among the four unique organic constituents of cells. They are responsible for a variety of important cell functions ranging from providing structural support to catalyzing biological reactions. They vary in shape, dynamic behavior, and localization. All of these together determine the specificity in their functions, but the question is how. The ultimate goal of the research conducted in this thesis is to answer this question. Two types of proteins are of particular interest. They include transmembrane proteins and protein assemblies. Using computer simulations with available experimental data to validate the simulation results, the research described here aims to reveal the structure and dynamics of proteins in their native-like environment and the indication on the mechanism of their functions. The first part of the thesis focuses on studying the structure and functions of transmembrane proteins. These proteins are consisted of transmembrane α-helices or β-strands, and each of the secondary structure elements adopts a unique orientation in the membrane following its local interactions. The structure of the entire protein is a collection of the orientations of these elements and their relative positions with respect to one another. These two basic aspects of membrane protein structure are studied in Chapter II and III. In Chapter II, efforts are given to determine the favorable orientation of a β-hairpin peptide, protegrin-1, in different lipid bilayers. The orientational preference results from the interplay between the protein and the surrounding lipid molecules. Chapter III is centered on revealing the structure and dynamics of caveolin-1 in DMPC bilayers. Caveolin-1 forms a re-entrant helix-turn-helix structure with two α-helices embedded in the membrane bilayer. The study shows that caveolin-1 monomer is rather dynamic and maintains its inserted conformation via both specific and non-specific protein-lipid interactions. To investigate the structural and dynamic impact on the function of a membrane protein, molecular dynamics simulations of the voltage-dependent anion channel are performed and the results are presented in Chapter IV. It is found in this chapter that the electrostatic interactions between charged residues on the channel wall facing the lumen are responsible for retarding the cation current, therefore giving the channel its anion selectivity. The second category of protein that is of interest in this thesis is the assembled protein complex, especially the ones that are highly symmetric. Actually, many membrane proteins belong to this category as well, but the study presented here in Chapter V involves simulations performed on a soluble protein complex, bacterioferritin B from Pseudomonas Aeruginosa. It is revealed by the simulations that the dynamic behavior of the protein is magnified by the symmetry and is tightly associated to its function

Membrane properties of structurally modified ceramides : effects on lipid lateral distribution and sphingomyelin-interactions in artificial bilayer membranes

Ceramides comprise a class of sphingolipids that exist only in small amounts in cellular membranes, but which have been associated with important roles in cellular signaling processes. The influences that ceramides have on the physical properties of bilayer membranes reach from altered thermodynamical behavior to significant impacts on the molecular order and lateral distribution of membrane lipids. Along with the idea that the membrane physical state could influence the physiological state of a cell, the membrane properties of ceramides have gained increasing interest. Therefore, membrane phenomena related to ceramides have become a subject of intense study both in cellular as well as in artificial membranes. Artificial bilayers, the so called model membranes, are substantially simpler in terms of contents and spatio-temporal variation than actual cellular membranes, and can be used to give detailed information about the properties of individual lipid species in different environments. This thesis focuses on investigating how the different parts of the ceramide molecule, i.e., the N-linked acyl chain, the long-chain sphingoid base and the membrane-water interface region, govern the interactions and lateral distribution of these lipids in bilayer membranes. With the emphasis on ceramide/sphingomyelin(SM)-interactions, the relevance of the size of the SMhead group for the interaction was also studied. Ceramides with methylbranched N-linked acyl chains, varying length sphingoid bases, or methylated 2N (amide-nitrogen) and 3O (C3-hydroxyl) at the interface region, as well as SMs with decreased head group size, were synthesized and their bilayer properties studied by calorimetric and fluorescence spectroscopic techniques. In brief, the results showed that the packing of the ceramide acyl chains was more sensitive to methyl-branching in the mid part than in the distal end of the N-linked chain, and that disrupting the interfacial structure at the amide-nitrogen, as opposed to the C3-hydroxyl, had greater effect on the interlipid interactions of ceramides. Interestingly, it appeared that the bilayer properties of ceramides could be more sensitive to small alterations in the length of the long-chain base than what was previously reported for the N-linked acyl chain. Furthermore, the data indicated that the SM-head group does not strongly influence the interactions between SMs and ceramides. The results in this thesis illustrate the pivotal role of some essential parts of the ceramide molecules in determining their bilayer properties. The thesis provides increased understanding of the molecular aspects of ceramides that possibly affect their functions in biological membranes, and could relate to distinct effects on cell physiology

Lipid Chemistry and Mechanical State of the Membrane Modulate Ion Channel Function

My research focused on voltage-dependent K+ (Kv) channels. Kv channels serve many di erent functions in di erent cells, but most notably underlie action potentials in electrically excitable cells, such as neurons and muscle (Hodgkin and Huxley, 1952, 1945). Kv channel gating is governed by the transmembrane voltage, they are therefore voltage-dependent switches for ionic current (Hille, 2001). Changes in the transmembrane voltage are sensed by the channel\u27s voltage sensor domains, which contain charged amino acids (most often arginines) called gating charges. Shortly before I started to work on my PhD project, the crystal structure of the eukaryotic Kv1.2 channel had been solved. This structure reinforced the idea that the voltage sensors are arranged as independent domains at the perimeter of a Kv channel facing the lipid membrane, thus exposing some of the gating charges to the lipid. The obvious question to ask at that time was, given the energetic penalty for placing charged amino acids inside the hydrophobic core of the membrane, how does the lipid membrane stabilize the arginine residues? By studying the recombinantlyexpressed arch al Kv channel KvAP in an arti cial membrane system that allowed me to create a de ned lipid environment, I could show that the lipid membrane provides an environment that is suitable for voltage sensors because the lipid\u27s phosphate groups serve as countercharges for the voltage sensor\u27s arginine residues. I came to the conclusion that a direct interaction between the arginine side chains and lipid phosphodiesters stabilizes the voltage sensor through multidentate hydrogen bonding. I suggested that the usage of positively charged amino acids in voltage sensors is an adaptation to the phospholipid composition of the cell membrane. Prompted by these results, I studied the gating properties of KvAP in di erent lipid systems and was able to derive the rst quantitative kinetic gating model for KvAP. I found that, unlike the well studied eukaryotic Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane in uences multiple gating transitions in the model, but most dramatically the rate of recovery from this inactivated state. I also showed that inhibition by the spider toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor\u27s return to the hyperpolarized conformation VSTx1 favors the inactivated state of KvAP. Aside from varying the chemical composition, I also studied how the mechanical state of lipid membranes in uences Kv channel gating. I found that Kv channels are mechanosensitive proteins and that a model in which membrane tension in uences a single parameter (the equilibrium constant governing pore-opening after the voltage sensors have moved) can account quantitatively for complex changes in voltagedependent gating, that are caused by the formation of tight lipid/glass seal in patch clamp recordings. The mechanical state of the membrane also governs the apparent a nity of spider toxins for Kv channels. This unexpected relationship between voltage sensor toxin a nity and the mechanical state of the membrane suggests that the toxin modi es the membrane mechanical forces experienced by the Kv channel. In summary, my thesis research describes how both the chemical and mechanical properties of lipid membranes regulate Kv channel function and pharmacology. These results demonstrate that the lipid membrane is not solely a passive solvent for membrane proteins, but that its composition and structure might be considered a source for functional diversity, enabling a membrane protein\u27s function to be tuned to the requirements of a particular cell type

Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

Over the past decade, the vast amount of information generated through structural and biophysical studies of GPCRs has provided unprecedented mechanistic insight into the complex signalling behaviour of these receptors. With this recent information surge, it has also become increasingly apparent that in order to reproduce the various effects that lipids and membranes exert on the biological function for these allosteric receptors, in vitro studies of GPCRs need to be conducted under conditions that adequately approximate the native lipid bilayer environment. In the first part of this review, we assess some of the more general effects that a membrane environment exerts on lipid bilayer-embedded proteins such as GPCRs. This is then followed by the consideration of more specific effects, including stoichiometric interactions with specific lipid subtypes. In the final section, we survey a range of different membrane mimetics that are currently used for in vitro studies, with a focus on NMR applications

Peptoid-modified Bicelles as Surrogate Cell Membranes for Membrane Protein Sensors and Analytics

Membrane-affiliated interactions are significant in understanding cell function, detecting biomarkers to diagnose disease, and in testing the efficiency of new therapeutic targets. Model membrane systems have been developed to study membrane proteins, allowing for stable protein structure and maintaining native activity. Bicelles, disc-shaped lipid bilayers created by combining long- and short-chain phospholipids, are the model membrane system of focus in this study. Bicelles are accessible from both sides and have a wide size range, which make them attractive for studying membrane proteins without affecting function. In this work, bicelles were functionalized with two peptoids to alter the edge and face chemistry. Peptoids are suitable for this application because of the large diversity of available side chain chemistries that can be easily incorporated in a sequence-specific manner. The peptoids sequence consist of three functional regions to promote insertion into the edge of bicelles. The insertion sequence at the C-terminus contains two alkyl chains and two hydrophobic, chiral aromatic groups that anchor into the bicelle edge or face. The facially amphipathic helix contains chiral aromatic groups on one side that interact with the lipid tails and positively charged groups on the other side, which interact with the lipid head groups. Thiol groups are included at the N-terminus to allow for determination of peptoid location in the bicelle. Bicelle morphology and size were assessed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Peptoid location in the bicelle was determined by attachment of gold nanoparticles, which confirm preferential incorporation of the peptoid into the bicelle edge or face. Results from this study show that peptoid-functionalized bicelles are a promising model membrane system. Specifically, the designed peptoids sequence were found to incorporate preferentially into the edges and faces of bicelles with 82% and 92% specificity, respectively. Additionally, the peptoid-functionalized bicelles are of similar size and morphology to non-functionalized bicelles. Potential applications would include customization to anchor in biosensors or facilitate interactions with specific membrane proteins or complexes

Striatin, a novel protein involved in the nongenomic/rapid action of steroids

Tese de doutoramento, Bioquímica (Bioquímica Clínica), Universidade de Lisboa, Faculdade de Ciências, 2013The cellular responses to steroids are mediated by two general mechanisms: genomic and rapid/nongenomic effects. Identification of the mechanisms underlying aldosterone’s rapid versus their genomic actions have been difficult to study and are not clearly understood. I explored the hypothesis that striatin is a critical intermediary of the rapid/nongenomic effects of aldosterone and that striatin serves as a novel link between the actions of the mineralocorticoid and estrogen receptors. In human and mouse endothelial cells, aldosterone promoted an increase in pERK that peaked at 15 minutes. Striatin is a critical mediator in this process as reducing striatin levels with siRNA technology prevented the rise in pERK levels. In contrast, reducing striatin did not significantly affect two well-characterized genomic responses to aldosterone. Down regulation of striatin with siRNA produced similar effects on estrogen’s actions – reducing nongenomic, but not the genomic actions investigated. Aldosterone, but not estrogen, increased striatin levels. When endothelial cells were pre-treated with aldosterone, the rapid/nongenomic response to estrogen on peNOS/eNOS ratio was enhanced and accelerated significantly. Importantly, pretreatment with estrogen did not enhance aldosterone’s nongenomic response on pERK. In conclusion, these results indicate that striatin is a novel mediator for both aldosterone’s and estrogen’s rapid and nongenomic mechanisms of action on pERK and peNOS, respectively, thereby providing evidence for a synergistic effect between the mineralocorticoid receptor and the estrogen receptor. Furthermore, these results suggest a unique level of interactions between steroids on the cardiovascular system that may have broad application for the treatment of cardiovascular diseases.Fundação para a Ciência e a Tecnologia (FCT, SFRH /BD/28601/2006 - POPH (QREN) - Formação Avançada), comparticipação do FSE e do MCTES e de subsídios do NHLBI/NIH USA: R01HL090632, R01HL094452 e R01HL09651

Dynamics and Interactions of Membrane Proteins

Membrane proteins are members of the class of proteins that perform their functions while being associated with a lipid bilayer. In the cell, they serve as transporters, receptors, anchors and enzymes. The domain organisation of these proteins suggests importance of lipid membrane and protein-lipid interactions for protein function. The requirement of a membrane mimic and the level of its resemblance to a native one for protein investigation makes the studies of membrane proteins a challenging project. My research work is focusing on the biophysical and biochemical studies of membrane proteins. This dissertation outlines two separate projects, each with their own challenges. Ras proteins are members of a superfamily of small GTPases that act as molecular switches that are involved in signal transduction pathways responsible for cell division and proliferation and, as one might guess, protein malfunction can lead to cancer. Recently, there have been a number of studies that suggest Ras protein dimerization on lipid membranes through protein-protein interactions between G- domains. On the basis of the results obtained from solution NMR and fluorescence polarization anisotropy studies, we concluded that the G-domain of the Ras protein by itself is not prone to dimerization. The result of this work was later confirmed by publications from other groups that performed studies in the presence of the lipid bilayer. NADPH-cytochrome P450 oxidoreductase (POR) is an integral membrane protein involved in an electron transport pathway transferring electrons from NADPH to cytochrome P450. The goal was achieved by application of lipid nanodisc technology, 13C-methyl extrinsic labeling coupled with Methyl-TROSY NMR technique that resulted in signals that showed differential sensitivity towards the redox state of the protein cofactors and conformational transitions of the protein. Moreover, results were obtained on a 600MHz instrument under protonated conditions. The goal of this project was the development of methodology to obtain structural data on a high-molecular weight protein associated with lipid nanodiscs in the presence of paramagnetic cofactors. Membrane proteins are challenging systems to research due to diverse interactions they experience on the membrane surface. In this dissertation I successfully utilized two approaches investigating this interactions: in my first project, I separately studied protein-protein interaction underlying the dimerization hypothesis, while in my second project I suggested the approach to explore conformational details and diverse interactions in a lipoprotein complex

Factors Influencing Opioid Receptor Signaling to Adenylyl Cyclase.

Opioids, such as morphine, signal through G protein-coupled receptors that are linked to adenylyl cyclase (AC)-inhibitory (Gi/o) heterotrimeric GTP-binding proteins. The transduction of signal from opioid receptor to G protein is dependent on the organization of proteins in the membrane and can be modulated by alterations in membrane lipids. This thesis provides evidence that mu-opioid receptors (MOR) and delta-opioid receptors (DOR) are differentially regulated by cholesterol. I have determined that cholesterol is required to stabilize MOR in a high-affinity conformation that couples to G proteins. Thus, cholesterol depletion attenuated the efficiency of MOR signaling to G proteins or AC. In contrast, DOR signaling was unaffected by removal of membrane cholesterol. Cholesterol depletion also attenuated the ability of MOR agonists to produce cellular adaptations leading to cAMP overshoot. This was true of heterologously expressed MOR in HEK293 cells, and endogenously expressed MOR in human neuroblastoma SH-SY5Y cells. Cholesterol may modulate MOR signaling by a direct interaction with the receptor or by formation of cholesterol-enriched membrane microdomains. Indeed, a portion of cell surface MOR was associated with markers of cholesterol-enriched membrane microdomains; however, these studies do not confirm that this association is responsible for the effect of cholesterol depletion on MOR signaling. In contrast, DOR was not associated with membrane microdomain markers, even after agonist treatment. MOR and DOR are thus likely not compartmentalized together within cholesterol-enriched membrane microdomains. Regardless, MOR and DOR in SH-SY5Y cells were observed to share a common pool of G proteins and AC, which resulted in occlusion of DOR agonist responses in the presence of maximally effective concentrations of MOR agonist. Moreover, this was not limited to MOR and DOR, as all Gi/o-coupled receptors endogenously expressed in SH-SY5Y cells were able to access a shared pool of AC. After chronic administration of MOR agonist, the addition of agonists to DOR, alpha2-adrenergic receptors (alpha2AR) and nociceptin/OFQ peptide receptors (NOPr) reduced the expression of MOR-mediated cAMP overshoot. Given the co-expression of alpha2AR and NOPr on MOR-containing neurons, the ability of agonists to these receptors to reduce MOR-mediated cAMP overshoot has implications in the treatment of opioid withdrawal symptoms.Ph.D.PharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/77937/1/elsawyer_1.pd

Studies of the exocytic snares involved in GLUT4 translocation

A specialised example of vesicular traffic is the translocation of the glucose transporter Glut4 from intracellular storage compartments to the plasma membrane of fat and muscle cells in response to insulin. In the basal state the majority of Glut4 is held intracellularly, sequestered away from the constitutively recycling endosomal pathway, in a proposed population of specialised Glut4 Storage Vesicles (GSVs). Insulin stimulates glucose transport into adipose cells by promoting the translocation of these GSVs to the plasma membrane where they fuse increasing the Glut4 levels at the cell surface and therefore significantly increasing facilitative glucose transport. The ability of insulin to stimulate glucose transport into muscle and adipose tissue, the main sites of glucose uptake, is central to the ability of insulin to regulate whole body glucose homeostasis, hi individuals with type 2 diabetes this ability of insulin to stimulate glucose transport is impaired. The incidence of type 2 diabetes is increasing rapidly and therefore understanding the molecular basis of insulin-stimulated glucose uptake is of fundamental importance. It has been over 25 years since the first evidence that insulin stimulation led to a translocation of glucose transport form an intracellular site to the plasma membrane of insulin responsive cells. Over this period of time significant advances have been made in the understanding of insulin-stimulated glucose uptake, both in the field of insulin signalling and Glut4 trafficking, however the intersection between these two processes is yet to be established. One major advance in the knowledge of Glut4 trafficking was the identification of the SNARE machinery involved in the fusion of GSVs to the cell surface. In eukaryotes all intracellular trafficking events are facilitated by a family of highly conserved proteins called SNAREs. GLUT4 containing vesicles are enriched in VAMP2, while the plasma membrane of adipocytes is enriched in syntaxin 4 and SNAP23, which together serve as a t-SNARE complex. In vitro these three SNAREs form a highly stable core complex and several studies show that these three proteins mediate the fusion of Glut4-containing vesicles with the plasma membrane. Whether this fusion event is regulated by insulin is yet to be established. The fusion process facilitated by SNARE proteins has been successfully reconstituted in vitro using recombinant proteins expressed in E. coli. Using the exocytic neuronal SNAREs in this assay it was demonstrated that SNAREpins, that is the complex formed between cognate sets of v- and t-SNAREs, are necessary and sufficient to fuse artificial membranes, ha Chapter 3 as the first step towards studying the regulation of fusion facilitated by syntaxin 4, SNAP23 and VAMP2, an in vitro fusion assay using these SNAREs was established. The three SNARE proteins were successfully expressed and purified from E.coli prior to reconstitution into synthetic liposomes that were subsequently analysed for fusion. The results of this assay show that these SNARE proteins in isolation are capable of fusing artificial membranes, a fact previously assumed but never definitively shown. (Abstract shortened by ProQuest.)