While conducting pilot studies into the usefulness of fusion to TELSAM polymers as a potential protein crystallization strategy, we observed novel properties in crystals of two TELSAM–target protein fusions, as follows. (i) A TELSAM–target protein fusion can crystallize more rapidly and with greater propensity than the same target protein alone. (ii) TELSAM–target protein fusions can be crystallized at low protein concentrations. This unprecedented observation suggests a route to crystallize proteins that can only be produced in microgram amounts. (iii) The TELSAM polymers themselves need not directly contact one another in the crystal lattice in order to form well-diffracting crystals. This novel observation is important because it suggests that TELSAM may be able to crystallize target proteins too large to allow direct inter-polymer contacts. (iv) Flexible TELSAM–target protein linkers can allow target proteins to find productive binding modes against the TELSAM polymer. (v) TELSAM polymers can adjust their helical rise to allow fused target proteins to make productive crystal contacts. (vi). Fusion to TELSAM polymers can stabilize weak inter-target protein crystal contacts. We report features of these TELSAM–target protein crystal structures and outline future work needed to validate TELSAM as a crystallization chaperone and determine best practices for its use.

The spatio-temporal attributes of intracellular calcium (Ca2+) transients activate various biological functions. These Ca2+ signaling events are triggered extracellularly through different stimuli and controlled intracellularly by the major Ca2+ storage organelle and by numerous Ca2+ pumps, channels, and Ca2+ binding proteins. Ca2+ transients can be significantly altered as a result of defects with signal modulation, leading to different diseases. Because of the fragility and intricacy of the Ca2+ signaling system, with the endo- and sarcoplasmic reticulum at the center, genetically-encoded Ca2+ probes that have been optimized for mammalian expression and fast kinetics are needed to observe global and local Ca2+ changes in different cells. Here, we first report the crystal structure determination of our genetically-encoded Ca2+ sensor CatchER which utilizes EGFP as the scaffold protein. Crystal structures of CatchER were resolved in the Ca2+-free, Ca2+-loaded, and gadolinium-loaded forms at 1.66, 1.20, and 1.78 Å, respectively. Analysis of all three structures established conformational changes in T203 and E222 produce the varying ratios of the neutral and anionic chromophore reflected in the absorbance spectrum where Ca2+ stabilizes the anionic chromophore and enhances the optical output. Since CatchER has miniscule fluorescence when expressed at 37˚C in mammalian cells, we enhanced its brightness by improving the folding at 37˚C, facilitating better chromophore formation. The resulting mutants are the CatchER-T series of Ca2+ sensors with CatchER-T’ having the most improvement in brightness at 37˚C. We also introduced the N149E mutation in the binding site to alter the Kd along with the brightness mutations. The resulting mutants were characterized and found to have weaker Kds compared to wild-type CatchER, similar quantum yields, and altered ratios of the neutral and anionic chromophore in the apo form. Then, CatchER-T’ was applied in situ to monitor Ca2+ changes globally in the ER/SR of C2C12, HEK293, and Cos-7 cells. A new construct consisting of CatchER-T’ and JP-45 was created to monitor local Ca2+ dynamics in the SR lumen of skeletal muscle cells. The results showed a difference between global and local SR Ca2+ release. We also examined the potential and spectroscopic properties to utilize some of our sensors in T cells to monitor the magnesium (Mg2+) flux in immune cells with faulty MagT1 receptors to understand the role of Mg2+ in the immune response.

Human Immunodeficiency Virus (HIV) has become a global pandemic with at least 25 million deaths and no cure. One of the most important targets to inhibit this virus is HIV-1 protease (PR), which is required to cleave the viral proteins needed for maturation of the virus after it invades and replicates in the host cell. There are nine protease inhibitors that are used in AIDS treatment. The virus loses susceptibility to these inhibitors by drug resistance due to mutations. The goal of the project is to examine the highly drug resistant HIV PR P51 in its complex with inhibitors. In this experiment we expressed and purified HIV PR P51 protein. We performed protein crystallization with inhibitors Tipranavir, Amprenavir, Darunavir, and Saquinavir to obtain the structure of the protease and the inhibitors in their complexes. Future analysis of the crystal structures will help with the development of successful therapeutic inhibitors.

Parasites of phylum Apicomplexa cause devastating diseases on a global scale. Toxoplasma gondii, the etiological agent of toxoplasmosis, and Plasmodium falciparum, the most virulent agent of human malaria, have the most substantial effects on human health and are the most widely studied. The success of these parasites is due in part to a sophisticated molecular arsenal that supports a variety of novel biological processes including a unique form of host cell invasion. Accessing the protective environment of the host cell is paramount to parasite survival and is mediated through an active invasion process: the parasite propels itself through a circumferential ring known as the moving junction (MJ) formed between its apical tip and the host cell membrane. The MJ ring is comprised of a parasite surface protein (AMA1) that engages a protein secreted by the parasite into the host cell and presented on the host cell surface (RON2). Thus, through an intriguing mechanism the parasite provides both receptor and ligand to enable host cell invasion. Prior to the studies described herein, the characterization of the AMA1-RON2 association was limited to low-resolution experiments that provided little insight into the functional and architectural details of this crucial binary complex. Towards elucidating the mechanism of AMA1-RON2 dependent invasion, I first structurally characterized T. gondii AMA1 bound to the corresponding binding region of RON2; analysis of the AMA1-RON2 interface along with biophysical data revealed an intimate association likely capable of withstanding the shearing forces generated as the parasite dives through the constricted MJ ring. To investigate the role of the AMA1-RON2 complex across genera, species and life-cycle stages, I next characterized the AMA1-RON2 complex from a distantly related genus within Apicomplexa (Plasmodium) and from a divergent pairing within T. gondii. By combining structural, biophysical and biological data, I was able to generate a detailed model describing the role of AMA1 and RON2 in MJ dependent invasion, which is currently supporting efforts to develop novel vaccines and cross-reactive small molecule therapeutics. / Graduate / 0487 / tonkin.ml@gmail.com

Human Immunodeficiency Virus (HIV) has become a global pandemic with at least 25 million deaths and no cure. One of the most important targets to inhibit this virus is HIV-1 protease (PR), which is required to cleave the viral proteins needed for maturation of the virus after it invades and replicates in the host cell. There are nine protease inhibitors that are used in AIDS treatment. The virus loses susceptibility to these inhibitors by drug resistance due to mutations. The goal of the project is to examine the highly drug resistant HIV PR P51 in its complex with inhibitors. In this experiment we expressed and purified HIV PR P51 protein. We performed protein crystallization with inhibitors Tipranavir, Amprenavir, Darunavir, and Saquinavir to obtain the structure of the protease and the inhibitors in their complexes. Future analysis of the crystal structures will help with the development of successful therapeutic inhibitors.

<div> <p>Guided by the continuous manufacturing paradigm shift in the pharmaceutical industry, the proposed thesis focuses on the implementation of an integrated continuous crystallization platform, the oscillatory baffled crystallizer (OBC), with real time process monitoring. First, by defining an appropriate operating regime with residence time distribution (RTD) measurements, a system can be defined that allows for plug flow operation while also maintaining solid suspension in a two-phase system. The aim of modern crystallization processes, narrow crystal size distributions (CSDs), is a direct result of narrow RTDs. Using a USB microscope camera and principal component analysis (PCA) in pulse tracer experiments, a novel non-contact RTD measurement method was developed using methylene blue. After defining an operating region, this work focuses on a specific process intensification technique, namely spherical crystallization.</p> <p>Used mainly to tailor the size of a final dosage form, spherical crystallization removes the need for downstream size-control based unit operations (grinding, milling, and granulation), while maintaining drug efficacy by tailoring the size of the primary crystals in the agglomerate. The approach for generating spherical agglomerates is evaluated for both small and large molecules, as there are major distinctions in process kinetics and mechanisms. To monitor the spherical agglomeration process, a variety of Process Analytical Technology (PAT) tools were used and the data was implemented for scale-up applications.</p> <p>Lastly, a compartmental model was designed based on the experimental RTD data with the intention of predicting OBC mixing and scale-up dynamics. Together, with validation from both the DN6 and DN15 systems, a scale independent equation was developed to predict system dispersion at different mixing conditions. Although it accurately predicts the behavior of these two OBC systems, additional OBC systems of different scale, but similar geometry should be tested for validation purposes.</p> </div> <br>

No description available.

We studied the usefulness of genetic fusion to TELSAM polymers as an effective protein crystallization strategy. We observed novel properties in crystals of two TELSAM-target protein fusions. TELSAM as a crystallization chaperone shows rapid crystallization when it's fused to target proteins and possibly with a greater propensity. Some TELSAM-target fusions crystallized more rapidly than the same target protein alone. TELSAM-target proteins can be crystallized at relatively low protein concentrations such as 0.1 mg/mL. TELSAM requires no TELSAM polymers touching one another in the crystal lattice in order to form well-diffracting crystals. This lack of crystal contacts has not been observed in previously reported TELSAM crystal structures. Flexible TELSAM-target protein linkers can allow target proteins to find productive binding modes against the TELSAM polymer. This study tested TELSAM linker lengths varying by the number of glycines, such as 2xGly, 4xGly, 6xGly, 8xGly, and 10xGly. Only TELSAM fused to UBA with 2 and 4 glycine linkers were crystalized. TELSAM polymers can adjust their helical rise to allow fused target proteins to make productive crystal contacts, and fusion to TELSAM polymers increases avidity to stabilize weak inter-target protein crystal contacts. In conclusion, we report features of TELSAM-target protein crystal structures and outline future work needed to validate TELSAM as a crystallization chaperone and define the best practices for its use.

Crystallization of proteins in the human body can lead to the development of diseases such as sickle cell anemia and cataract. Understanding protein crystallization can give insight into such diseases. Furthermore, protein crystallization is necessary for protein structure resolution. This is important since resolution of protein structure is the first step towards establishing structure/function relations, and possibly towards performing specific structural modifications that may change the function in desirable directions. Another important application of protein crystallization is in downstream processing in the pharmaceutical industry where it is used for separation and as a final purification step. The present study increases knowledge of interactions between protein molecules during crystallization and hence the crystallization process. Crystallization of proteins in the human body can lead to the development of diseases such as sickle cell anemia and cataract. Understanding the processes involved in protein crystallization can help us gain a better understanding of such diseases. Crystallization of human gamma D-crystallin (HGD) and apoferritin, two proteins found in the lens, was studied in relation to cataract formation. Crystallization of both proteins was studied in the presence of divalent cations which are found at elevated concentrations in cataractous lenses. Results indicate that the divalent cations studied enhance crystallization of these proteins. A thermodynamic property, the osmotic second virial coefficient, was measured in protein solutions and its value was correlated with the occurrence of crystallization. It was found that the second virial coefficient successfully predicted crystallization of both proteins. A new method was developed for indirect measurement of the second virial coefficient using dynamic light scattering. This new method is more robust and efficient than the traditional static light scattering method. Finally the ability of solubilizers to prevent crystallization in HGD solutions was studied. A commercial solubilizer, NDSB-201, was found to increase the energy barrier to nucleation. Although this did not prevent crystallization, it resulted in fewer and smaller crystals being obtained. The naturally occurring alpha A-crystallin was a superior solubilizer to NDSB-201, as it suppressed aggregation and prevented crystallization of HGD under conditions for which NDSB-201 did not. The findings in the present study provide insight into the processes by which protein crystallization occurs and hence into diseases associated with protein crystallization. The findings in the present study provide insight into the processes by which protein crystallization occurs. Using the second virial coefficient to assess whether a protein will crystallize out of solution, approaches for retardation and prevention of protein crystallization, and implications for future research, are discussed.

<p>X-ray and neutron crystallography are the predominant methods for obtaining atomic-scale information on bimolecular macromolecules. Despite the success of these techniques, generating well diffracting crystals critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. </p><p>The fields of structural biology and soft matter have independently sought out fundamental principles to rationalize protein crystallization. Yet the conceptual differences and limited overlap between the two disciplines may have prevented a comprehensive understanding of the phenomenon to emerge. Part of this dissertation focuses on computational studies of rubredoxin and human uniquitin that bridge the two fields.</p><p>Using atomistic simulations, the protein crystal contacts are characterized, and patchy particle models are accordingly parameterized. Comparing the phase diagrams of these schematic models with experimental results enables the critical review of the assumptions behind the two approaches, and reveals insights about protein-protein interactions that can be leveraged to crystallize proteins more generally. In addition, exploration of the model parameter space provides a rationale for several experimental observations, such as the success and occasional failure of George and Wilson's proposal for protein crystallization conditions and the competition between different crystal forms.</p><p>These simple physical models enlighten the connection between protein phase behavior and protein-protein interactions, which are, however, remarkably sensitive to the protein chemical environment. To help determine relationships between the physico-chemical protein properties and crystallization propensity, statistical models are trained on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis.</p><p>To conclude, the behavior of water in protein crystals is specifically examined. Water is not only essential for the correct functioning and folding of proteins, but it is also a key player in protein crystal assembly. Although water occupies up to 80% of the volume fraction of a protein crystal, its structure has so far received little attention and it is often overly simplified in the structural refinement process. Merging information derived from molecular dynamics simulations and original structural information provides a way to better understand the behavior of water in crystals and to develop a method that enriches standard structural refinement.</p> / Dissertation