Protein interactions occurring through biomolecular interfaces play an important role in the circle of life. These interactions are responsible for cellular function, including RNA transcription, protein translation, cell division and cell death among many others. There are different types of interactions based on the strength and the duration of the association. Transient interactions govern most steps of the cellular metabolism, where the associations between two or more molecules are responsive to environmental cues. Among the participants of transient interactions, intrinsically disordered proteins are employed in signaling and other regulatory events within the cell. These proteins exhibit allosteric regulation and gain secondary structure when they bind other proteins or small molecules. In this doctoral thesis work, the biochemical and biophysical principals governing protein associations are investigated and using protein engineering tools, novel biomolecular interfaces are engineered, with potential applications in different areas of biotechnology. The first part of the thesis (Chapter 2) focuses on the investigation of supramolecular enzyme association among tricarboxylic acid cycle enzymes, specifically between citrate synthase and mitochondrial malate dehydrogenase. In this study, the interactions between these enzymes are examined, both among their natural and synthetically produced recombinant versions. In addition, mutational analysis of the amino acid residues at the complex interface was performed to explore the importance of the positively charged patch connecting the active sites of the enzymes. It was discovered that the channeling of the negatively charged intermediate is severely impaired upon mutation of surface residues contributing to the electrostatic channeling. This work provides an important insight into understanding the coupled reaction-transport systems and metabolon formation in general. In addition, it constitutes a great example for substrate channeling in leaky systems, which are relevant to most biological processes. The next section of the thesis (Chapter 3) focuses on an intrinsically disordered peptide, the β-roll. This peptide is isolated from the Block V repeats-in-toxin (RTX) domain of adenylate cyclase from Bordetella pertussis. It is disordered in the absence of calcium and it folds into a β-roll secondary structure composed of two parallel β-sheet faces upon binding to calcium ions. This way, the peptide can transition between its unfolded state and the β-roll structure in a reversible way. We have utilized the allosteric regulation of this domain as a tool to engineer new protein interfaces. In its folded state, the peptide has two faces, serving as binding surfaces available for interaction with other proteins. Our work involved the alteration of the residues, which form these faces upon calcium binding, via combinatorial protein design techniques. The potential of this peptide is evaluated as a cross-linking domain for hydrogel formation. By rationally engineering the two faces of the folded β-roll to contain leucine residues, we have created hydrophobic interfaces, serving as environmentally-responsive cross-linking domains. When there is no calcium, the β-roll domains remain unstructured, delocalizing the leucine rich patches. After calcium binding, the β-rolls fold and the leucine rich faces are exposed creating a hydrophobic driving force for self-assembly. This way, we showed that the β-roll peptide can function as a biomaterials building block capable of proteinaceous hydrogel formation, only in the presence of calcium. The next study (Chapter 4) demonstrates the utilization of this peptide as an alternative scaffold for biomolecular recognition applications. A library of mutant β-rolls was constructed by randomizing the amino acid residues on one of the β-sheet forming faces. Mutant peptides demonstrating an affinity for hen egg white lysozyme were selected, which was chosen as a model target molecule. The thermodynamic parameters of the interactions between the β-roll mutants and the lysozyme were quantified. Upon performing further protein engineering (e.g. concatenation of the single mutants on the DNA level), a mutant with mid-nanomolar affinity was identified. Affinity chromatography experiments showed that this mutant was capable of capturing the target, in the presence of calcium. The captured target was easily released upon removal of the calcium ions. The reversibility of the calcium binding allowed the engineered molecular interface to be controllable. Throughout this study, the β-roll peptide was explored as an allosterically-regulated protein switch for on/off biomolecular recognition, which can be mediated by simply changing the calcium concentration, allowing control over the binding behavior between molecules. The last part of the thesis (Chapter 5) expands on the calcium dependent network formation study. A hydrogel construct was genetically built by fusing the cross-linking β-roll domain and the lysozyme binding β-roll mutant, resulting in a smart biomaterial with dual-functionality. The network-assembly and target capture functions of this construct were tested by various assays including hydrogel erosion experiments. This allosterically-regulated biomaterial exhibited promising results, where calcium-dependent lysozyme entrapment within the assembled network and lysozyme capture on the hydrogel surface were demonstrated. The work presented in this thesis demonstrates different approaches to understand and engineer molecular interfaces in both natural and recombinant systems. In the future, these approaches and the knowledge gained from these studies can be further built upon for different biotechnological applications and can also be applied to other synthetic systems.

Novel biocatalysts for the enantioselective reduction of imines and reductive amination of a broad range of carbonyl compounds have been developed. Unlike other imine reductases (IREDs), the IRED from Amycolaptosis orientalis (AoIRED) features an aprotic "catalytic" residue Asn171 and as such became an interesting candidate for detailed mechanistic, specificity and stereoselectivity studies. AoIRED has been shown to be an efficient catalyst for the enantioselective reduction of imines and iminium ions to yield the corresponding chiral amines in high conversions and good to excellent enantioselectivity. The enzyme exhibits unusual stereoselective properties, displaying a selectivity switch for structurally similar substrates and in certain cases for the same substrate depending on the age of the enzyme. Mutagenesis studies have highlighted important residues that may play key roles in the substrate specificity and stereoselectivity of the enzyme. The reductive aminase from Aspergillus oryzae (AspRedAm) is a multifunctional catalyst that efficiently catalyses i) the reductive coupling of carbonyl compounds and amine nucleophiles, ii) the enantioselective reduction of prochiral cyclic and preformed imines or iii) the oxidative deamination of amines towards kinetic resolution of racemic amines. Detailed kinetic studies have led to the construction of a kinetic model/mechanism and based on structure guided investigation of conserved active site residues, a putative catalytic mechanism has been proposed. It has also been possible to engineer wild-type AspRedAm for improved stereoselectivity as well as to invert the enzyme's enantioselectivity towards a range of substrates. Using AspRedAm as a catalyst, efficient systems have been developed that allow the kinetic resolution of several racemic amines. This thesis has been organised into separate chapters each addressing a specific theme. Chapter 1 gives an overview of recent advances in the field of amine biocatalysis with emphasis on biocatalytic imine reduction and reductive amination; it also outlines the objectives of this project. Chapter 2 describes methods and materials used in these studies while Chapters 3-7 present and discuss results from different projects that constitute the work in this thesis. Initial discovery and characterisation studies of IREDs are described in Chapter 3. Chapter 4 describes detailed characterisation of AoIRED with particular emphasis on stereoselectivity and synthetic applicability while Chapter 5 presents and discusses results from the study of the reductive aminase (AspRedAm) from Aspergillus oryzae. Chapters 6 and 7 respectively describe the engineering of AoIRED and AspRedAm, and the application of AspRedAm in kinetic resolution of racemic amines. The results from these chapters have been summarised and discussed in Chapter 8 and recommendations for future directions in this field have been offered.

Electrostatic interaction has long been proposed to be an important factor for stabilizing protein. Charge-charge interaction may especially be important to the thermostability of protein, as having more surface electrostatic interactions is one of the common structural features found in thermophilic proteins when compared to their mesophilic homologues. In order to quantitatively investigate the electrostatic contribution to protein stability, two complementary approaches, namely the double mutant cycle approach and pKa shift approach, were carried out. / In the double mutant cycle approach, the coupling free energies of two salt bridges (E6/R92 and K46/E62) and one a long range ion pair (E90/R92) were estimated by using circular dichroism, to find out the thermodynamic parameters of the protein model Thermococcus celer L30e and its charge-to-neutral mutants. It was found that the coupling free energy was temperature independent and was about 3 kJ mol-1 per salt bridge. By using a novel analysis of double mutant cycle of DeltaC p, it was also found that the interaction of salt bridge plays an important role in the reduction of DeltaCp. The temperature independency of coupling free energy and the effect of reducing DeltaCp could explain the general observation very well that thermophilic proteins have highly up-shifted protein stability curves is due to its elevated electrostatic interactions when compared with their mesophilic homologs. / In the pKa shift approach, the native state pKa values of acidic residues were obtained by fitting the side chain carboxyl 13C chemical shifts to microscopic model or global fitting of titrational event (GloFTE), whereas the denatured state pKa values were obtained by conventional pH titration of terminal protected 5-residue glycine-based model peptide. It was found that the surface charge-charge interactions, either attractive or repulsive, were strong and complicated because of the high surface charge density of T. celer L30e. However, the fact that most of the acidic residues have significantly downshifted native state pK a values indicated the surface charge distribution of T. celer L30e is optimized for stabilizing the protein. In addition, we have shown that temperature has negligible effect on pKa values in both native state and denatured state, therefore temperature can only marginally amplify the stabilizing effect in linear manner. / To overcome the unwanted crystallization problem of wild-type T. celer L30e in the low ionic strength neutral pH NMR conditions, which were essential for the pKa shift approach, a quintuple Arg-to-Lys variant was designed to dramatically improve the crystalline solubility, while the surface charges, as well as the structural, thermodynamic, and electrostatic properties, were conserved. It has also shown that electrostatic interaction played a critical role in crystallization at low ionic strength conditions, and arginine residue was especially important in crystal packing because of its high ability of forming salt bridges and hydrogen bonds. / Wild-type T. celer L30e has also shown to have no observable residual structure in the guanidine HC1-induced denatured state, indicating that denatured state of T. celer L30e should not have large effect on the overall protein stability. / Chan, Chi Ho. / Adviser: Kam Bo Wong. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 202-218). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

A L-asparaginase (EC 3.5.1.1) é uma enzima importante para o tratamento da leucemia linfoblástica aguda (LLA), neoplasia mais frequente em crianças e adolescentes. A L-asparaginase hidrolisa a L-asparagina resultando em ácido aspártico e amônio, impedindo que as células tumorais utilizem esse aminoácido para síntese proteica, ocasionando a morte celular apoptótica. Atualmente a enzima é obtida a partir de Escherichia coli e Erwinia chrysanthemi; no entanto, ambas as formulações estão associadas a um alto índice de efeitos adversos que comprometem a evolução e eficácia do tratamento. A levedura Saccharomyces cerevisiae tem o gene ASP1 responsável pela produção de L-asparaginase 1 (Sc_ASPase1) que tem sido pouco estudada. Para elucidar as características de Sc_ASPase1 nós expressamos a proteína em E. coli BL21(DE3) e a purificamos por cromatografia de afinidade. Sc_ASPase1 tem uma atividade especifica de 195,4 U/mg para L-asparagina e de 0,36 U/mg para L-glutamina, e um comportamento alostérico com um K0.5 de 75 µM para L-asparagina. Por meio de mutação sitio dirigida demonstramos a importância dos resíduos Thr64-Thy78-Th141-Lys215 para a catálise. As isoformas mutantes da proteína A331D, K335E, Y243S, S301N e ΔG77 não apresentaram melhoria nos parâmetros cinéticos ou atividade específica. Construímos e clonamos Sc_ASPase1 com a deleção dos primeiros 52 aminoácidos, porém nas condições testadas a proteína foi expressa na forma insolúvel. Demonstramos que Sc_ASPase1 possui potencial antineoplásico, pois com 10 U/mL de enzima foi capaz causar a 85% de mortalidade da linhagem leucêmica MOLT-4. Na mesma concentração, a enzima de E. coli é capaz de matar 95% de células dessa mesma linhagem. / L-Asparaginase (EC 3.5.1.1) is an important enzyme for the treatment of acute lymphoblastic leukemia (ALL), the most common malignancy in children and adolescents. L-asparaginase hydrolyzes L-asparagine resulting in ammonium and aspartic acid, preventing tumor cells of using such amino acid for protein synthesis, leading to apoptotic cell death. Currently, the enzyme is obtained from Escherichia coli and Erwinia chrysanthemi; however, both formulations are associated with a high incidence of side effects that compromise the progress and effectiveness of treatment. The yeast Saccharomyces cerevisiae has ASP1 gene responsible for the production of L-asparaginase 1 (Sc_ASPase1) that has been poor studied. To elucidate the characteristics of Sc_ASPase1, we expressed the protein in E. coli BL21 (DE3) cells and purified it by affinity chromatography. Sc_ASPase1 has a specific activity of 195.4 U/mg for L-asparagine and 0.36 U/mg for L-glutamine, and an allosteric behavior with a K0.5 of 75 µM for L-asparagine. Through site directed mutation, we demonstrated the importance of Thr64-Thy78-Th141-Lys215 residues for catalysis. The mutant protein isoforms A331D, K335E, Y243S, S301N and ΔG77 showed no improvement in kinetic parameters or specific activity. We build and cloned Sc_ASPase1 with the deletion of the first 52 amino acids, but under the conditions tested the protein was expressed in insoluble form. Sc_ASPase1 have demonstrated potential antineoplastic activityc, since 10 U/mL of enzyme lead to 85% of mortality in leukemia cell line MOLT-4. At the same concentration, the E. coli enzyme kills 95% of the cells of the same line.

PSD-95/Dlg/ZO-1 (PDZ) domain - PDZ binding motif (PBM) interactions have been one of the most well studied protein-protein interaction systems through biochemical, biophysical and high-throughput screening (HTS) strategies. This has allowed us to understand the mechanism of individual PDZ-PBM interactions and the re-engineering of PBMs to bind tighter or to gain or lose certain specificity. However, there are several thousand native PDZ domains whose biological ligands remain unknown. Because of the low sequence identity among PDZ domain homologues, promiscuous binding profiles (defined as a PDZ domain that can accommodate a set of PBMs or a PBM that can be recognized by many PDZ domains), and context-dependent interaction mechanism, we have an inadequate understanding of the general molecular mechanisms that determine the PDZ-PBM specificity. Therefore, predicting PDZ specificity has been elusive. In addition, no de novo PBM ligand or artificial non-native PDZ domain have been successfully designed. This reflects the general challenges in understanding the general principles of PDZ-ligand interactions, namely that they are context-dependent, exhibit weak binding affinity, narrow binding energy range, and larger interaction surface than other protein-ligand interactions. Together, PDZ domains make good model systems to investigate the fundamental principles of protein-protein interactions with a wide spectrum of biomedical implications. My studies suggest that understanding PBM specificity with the set of structural positions forming the binding pocket can connect sequence, structure and function of a PDZ domain in a general context. They also suggest that this way of understanding the specificity will shed light on prediction and engineering of specificity rationally. Structural analysis on most of the available PDZ domain structures was established to support the principle (Chapter I). The principle was tested against two different types of PBM; C-terminal PBM (Chapter II) and internal PBM (Chapter III), and shown to support better understanding and design of PDZ domain specificity. We further applied the principle to design de novo PDZ domains, and the preliminary data hints that it is optimistic to engineer PDZ domain specificity (Appendix A and B).

Proteins are large macromolecules that play important roles in nature. With the development of modern molecular biology techniques, protein engineering has emerged as a useful tool and found many applications in areas ranging from food industry, environmental protection, to medical and life science. Biomimetic membrane incorporates biological elements, such as proteins, to form membranes that mimic the high specificity and conductance of natural biological membranes. For any application involving the usage of proteins, the first barrier is always the production of proteins with sufficient stability, and the incorporation of proteins into the artificial matrix. This thesis contains two major parts, the first part is focused on the development and testing of method to immobilize active enzymes. The second part is devoted to study the degradation of membrane proteins in E. coli cells. In the immobilization study, Pyrophosphatase (PpaC) was chose as a model enzyme. A dual functional tag consist of histidine and methionine has been developed, in which histidine is used for purification while methionine is metabolically replaced with azidohomoalanine (AHA) for immobilization. We found that the addition of the tag and the incorporation of AHA did not significantly impair the properties of proteins, and the histidine–AHA tag can facilitate protein purification, immobilization, and labeling. This tag is expected to be useful in general for many proteins. Degradation of soluble protein has been well characterized, but the membrane protein degradation process remains elusive. SsrA tag is a well-known recognition sequence for soluble protein degradation, which marks prematurely terminated protein products translated from damaged mRNA. SsrA tagged membrane proteins was found to be substrate of a cytosolic protease complex ClpXP, which mediated complete degradation.

Naturally occurring enzymatic pathways enable highly specific, rapid thiophenic sulfur cleavage occurring at ambient temperature and pressure, which may be harnessed for the desulfurization of petroleum-based fuel. One pathway found in bacteria is a four-step catabolic pathway (the 4S pathway) converting dibenzothiophene (DBT), a common crude oil contaminant, into 2-hydroxybiphenyl (HBP) without disrupting the carbon-carbon bonds. 2’-Hydroxybiphenyl-2-sulfinate desulfinase (DszB), the rate-limiting enzyme in the enzyme cascade, is capable of selectively cleaving carbon-sulfur bonds. Accordingly, understanding the molecular mechanisms of DszB activity may enable development of the cascade as industrial biotechnology. Based on crystallographic evidence, we hypothesized that DszB undergoes an active site conformational change associated with the catalytic mechanism. Moreover, we anticipated this conformational change is responsible, in part, for enhancing product inhibition. Rhodococcus erythropolis IGTS8 DszB was recombinantly produced in Escherichia coli BL21 and purified to test these hypotheses. Activity and the resulting conformational change of DszB in the presence of HBP were evaluated. The activity of recombinant DszB was comparable to the natively expressed enzyme and was competitively inhibited by the product, HBP. Using circular dichroism, global changes in DszB conformation were monitored in response to HBP concentration, which indicated that both product and substrate produced similar structural changes. Molecular dynamics (MD) simulations and free energy perturbation with Hamiltonian replica exchange molecular dynamics (FEP/λ-REMD) calculations were used to investigate the molecular-level phenomena underlying the connection between conformation change and kinetic inhibition. In addition to the HBP, MD simulations of DszB bound to common, yet structurally diverse, crude oil contaminates 2’2-biphenol (BIPH), 1,8-naphthosultam (NTAM), 2-biphenyl carboxylic acid (BCA), and 1,8-naphthosultone (NAPO) were performed. Analysis of the simulation trajectories, including root mean square fluctuation (RMSF), center of mass (COM) distances, and strength of nonbonded interactions, when compared with FEP/λ-REMD calculations of ligand binding free energy, showed excellent agreement with experimentally determined inhibition constants. Together, the results show that a combination of a molecule’s hydrophobicity and nonspecific interactions with nearby functional groups contribute to a competitively inhibitive mechanism that locks DszB in a closed conformation and precludes substrate access to the active site. Limitations in DszB’s potential applications in industrial sulfur fixation are not limited to turnover rate. To better characterize DszB stability and to gain insight into ways by which to extend lifetime, as well as to pave the way for future studies in inhibition regulation, we evaluated the basic thermal and kinetic stability of DszB in a variety of solvation environments. Thermal stability of DszB was measured in a wide range of different commercially available buffer additives using differential scanning fluorimetry (DSF) to quickly identify favorable changes in protein melting point. Additionally, a fluorescent kinetic assay was employed to investigate DszB reaction rate over a 48 hr time period in a more focused group of buffer to link thermal stability to DszB life-time. Results indicate a concerningly poor short-term stability of DszB, with an extreme preference for select osmolyte buffer additives that only moderately curbed this effect. This necessitates a means of stability improvement beyond alteration of solvation environment. To this end, a more general investigation of glycosylation and its impact on protein stability was performed. Post-translational modification of proteins occurs in organisms from all kingdoms life, with glycosylation being among the most prevalent of amendments. The types of glycans attached differ greatly by organism but can be generally described as protein-attached carbohydrate chains of variable lengths and degrees of branching. With great diversity in structure, glycosylation serves numerous biological functions, including signaling, recognition, folding, and stability. While it is understood that glycans fulfill a variety of important roles, structural and biochemical characterization of even common motifs and preferred rotamers is incomplete. To better understand glycan structure, particularly their relevance to protein stability, we modeled and computed the solvation free energy of 13 common N- and O-linked glycans in a variety of conformations using thermodynamic integration. N-linked glycans were modeled in the β-1,4-linked conformation, attached to an asparagine analog, while O-linked glycans were each modeled in both the α-1,4 and β-1,4-linked conformations attached to both serine and threonine analogs. Results indicate a strong preference for the β conformation and show a synergistic effect of branching on glycan solubility. Our results serve as a library of solvation free energies for fundamental glycan building blocks to enhance understanding of more complex protein-carbohydrate structures moving forward.

Many histidine-rich sites in proteins bind transition metal ions such as Zn2+, natively. Such sites can encourage proper protein folding or allow access to enzymatic capabilities such as hydrolysis. Ru(II) and Tc(I) also bind to aromatic amines providing access to unique chemistries not observed in biology. Ru(II) complexes have shown efficacy in fighting cancer and catalysis, while 99mTc complexes are used in radio-imaging. To incorporate such metal-ions' activities into proteins, several mutants have been designed to bind Zn2+, Ru2+, and Tc1+ by introducing three histidines onto their surfaces. The first design, Z0, utilized a chimeric approach by substituting a turn in engrailed homeodomain for the superimposable Zn2+-binding loop of astacin. In the second design, 3HT-C, three histidine residues were incorporated into the N-terminus of the Trp-cage. The final scaffold, ubiquitin, was used to make two mutants: 3HIU with a 3-histidine containing loop inserted between residues 9 and 10, and 3HPU with 3-histidine point mutations near residues 35-38. Z0 proved unstable due to incorporation of a hydrophobic patch onto its surface and was not able to be isolated in sufficient quantities for study. However, the other proteins were stable and soluble. Zn2+-binding by 3HT-C was investigated by intrinsic tryptophan fluorescence quenching, circular dichroism, and RP-HPLC. Binding by 3HIU and 3HPU was studied by CD. All designed proteins bind to Zn2+ with Kd values in the micromolar range. 3HIU and 3HPU were further studied for their ability to bind Ru(tacn)2+ complexes. While addition of Ru-complexes caused oligomerization to various extents depending upon reaction conditions, homogeneous Ru-protein monomers were purified by a combination of size-exclusion, cation-exchange and immobilized-metal affinity chromatographies. Ru-binding was confirmed by ESI-MS, and structural integrity was investigated by CD. Results indicate that Ru(tacn)2+ complexes can be bound to surface binding sites in proteins without disruption of structure, opening the door for the study of catalysis in a protein context. 99mTc(CO)3+-binding studies were performed with 3HIU by RP-HPLC. This protein binds Tc+ and resists substitution by free L-histidine, suggesting that peptidic Tc-binding tags could be designed with this approach to readily incorporate the radionuclide into any protein expression system.

PHAs are a class of biopolymers consisting of (R)-3-hydroxy-fatty acids and are produced by the majority of eubacteria and some archaeal bacteria as carbon storage material. In general, PHA is synthesised when a carbon source is available in excess while another essential nutrient is limited. The key enzyme of PHA biosynthesis, the PHA synthase, catalyses the polymerisation of the substrate (R)-3-hydroxyacyl-CoA to PHA accompanied by the release of coenzyme A. PHA is stored intracellularly as inclusions, the so-called PHA granules. When the external carbon source becomes exhausted, bacteria can metabolise these carbon inclusions by degradation of the polymer. PHA granules are water-insoluble, spherical inclusions of approximately 50-500 nm in diameter which consist of a hydrophobic polyester core surrounded by a phospholipid layer with embedded and attached proteins. One could consider isolated PHA granules as bio-beads due to their structure and size. In this study we tested if the PHA synthase can be used as an anchor molecule in order to display proteins of interest at the PHA granule surface. Furthermore, these modified PHA granules were analysed for their potential applicability as bio-beads in biotechnological procedures. The concept of using the PHA synthase as granule-anchoring molecule for display of proteins of interest was established by the functional display of the ß- galactosidase at PHA granules. This “proof of concept” was followed by the display of biotechnologically more interesting proteins. The IgG binding domain of protein A as well as streptavidin, which is known for its biotin binding ability, were fused to the PHA synthase, respectively, and therefore localised at the PHA granule surfaces during PHA granule assembly, resulting in functional bio-protein A -beads and bio-streptavidin-beads. Moreover, their applicability in biotechnological assays was demonstrated. Recently, we fused the green fluorescent protein (GFP) to the PHA synthase and demonstrated that the PHA granule assembly does not start randomly distributed in the cytoplasm but occurred localised at or near the cell poles. To further investigate if the localisation of the PHA granule formation process is due to polar positional information inherent to the PHA synthase, different mutated versions of the PHA synthase of Cupriavidus necator were created and analysed for a potential alteration in localisation. Furthermore, the phasin protein PhaP1 of C. necator was fused to HcRed, a far-red fluorescent protein, and localisation studies were accomplished when the fusion protein was expressed under different conditions in Escherichia coli.

Environmental signals are sensed by membrane-spanning receptors that communicate with the cell interior. Bacterial chemoreceptors modulate the activity of the CheA kinase in response to binding of small ligands or upon interaction with substrate-bound periplasmic-binding proteins. The mechanism of signal transduction across the membrane is a displacement of the second transmembrane domain (TM2) a few angstroms toward the cytoplasm. This movement repositions a dynamic transmembrane helix relative to the plane of the cell membrane. The research presented in this dissertation investigated the contribution of TM2-membrane interactions to signaling by the aspartate chemoreceptor (Tar) of Escherichia coli. Aromatic residues that reside at the cytoplasmic polar-hydrophobic membrane interface (Trp-209 and Tyr-210) were found to play a significant role in regulating signaling by Tar. These interactions were subsequently manipulated to modulate the signaling properties of Tar. The baseline signaling state was shown to be incrementally altered by repositioning the Trp-209/Tyr-210 pair. To our knowledge, this is the first example of harnessing membrane-protein interactions to modulate the signal output of a transmembrane receptor in a controlled and predictable manner. Potential long-term applications include the use of analogous mutations to elucidate two-component signaling pathways, to engineer the signaling parameters of biosensors that incorporate chemoreceptors, and to predict the movement of dynamic transmembrane helices in silico.