The p53 protein is a critical tumor suppressor that is mutated in over half of all human cancers. It plays essential roles in maintaining genomic integrity by modulating the cellular response to various types of genotoxic stress. Associating with over 270 proteins to date, one of the mechanisms pivotal to p53’s multifaceted activities is protein-protein interactions. As to how most of these molecules bind to and affect p53 function remains unclear. Here we present a combined structural and biophysical approach to study three p53-interacting partners: BRCA1, IFI16 and p53 affinity reagent in an attempt to elucidate the basis of how these proteins recognize, bind to and alter p53’s biochemical functions. We have biophysically characterized the central region of BRCA1 and examined how it acts as a disordered scaffold to mediate association with p53 and other proteins. Having a putative role as a tumor suppressor, we have determined the crystal structures of the HIN-A and HIN-B domains of IFI16 and find that they interact with the C-terminus and DNA-binding core domain of p53, respectively, and enhance the DNA binding and transactivation activities of p53. Most cancer hot spot mutations of p53 are localized in the core domain and are thermally destabilized. Attaining molecules that stabilize the p53 fold has therefore been regarded as an attractive approach for cancer therapy. Lastly, using a phage-displayed library, evidence is presented to demonstrate a proof-of-principle for generating synthetic affinity reagents to potentially restore the function of tumor-derived p53 core domain mutants.

Single-molecule fluorescence spectroscopy techniques such as Fluorescence Correlation Spectroscopy (FCS) and single-molecule Förster Resonance Energy Transfer (smFRET) not only possess an unprecedented high sensitivity but also have high temporal and spatial resolution. Therefore, they have an immense potential both in investigation of fundamental biological principles and in clinical applications. FCS analyses are based on both theoretical approximations of the beam geometry and assumptions of the underlying molecular processes. To address the accuracy of analysis, firstly the experimental conditions that should be fulfilled in order to obtain reliable physical parameters are discussed and the input parameters are carefully controlled accordingly to demonstrate the performance of FCS measurements on our home-built confocal multiparameter photon-counting microscope in several in vitro and in-vivo applications. Secondly, we performed a comprehensive FCS analysis of rhodamine family of dyes to evaluate the validity of assigning the correlation relaxation times to the time constant of conformational dynamics of biomolecules. While it is the common approach in literature our data suggests that conformational dynamics mainly appear in the correlation curve via modulation of the dark states of the fluorophores. The size and shape of the folded, unfolded and chemically-denatured states of the N-terminal Src-homology-3 of downstream of receptor kinases (DrkN SH3) were investigated by FCS and smFRET burst experiments. Based on the data, we conclude that a considerable sub-population of the denatured protein is in a closed loop state which is most likely formed by cooperative hydrogen bonds, salt bridges and nonpolar contacts. As a clinical application, we developed and characterized an ultrasensitive capillary electrophoresis method on our multiparameter confocal microscope. This allowed us to perform Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with about 500 times better sensivity than a commercial instrument. Quite remarkably, we were able to analyze samples of cell lysate down to the contents of a single cell.

Farber disease is a rare lysosomal storage disease (LSD) caused by a deficiency of acid ceramidase (AC). Patients show a classic triad of symptoms including subcutaneous granulomas, laryngeal involvement and painful swollen joints. The most common and severe form has neurological manifestations and patients typically die by the age of two. Current treatment consists of symptomatic supportive care and allogeneic bone marrow transplantation (BMT). However, BMT has shown limited success. Gene therapy has previously been shown to be a promising treatment strategy for monogenetic diseases and has the potential to treat the underlying cause of the disease. Presented here is the first report of in vivo testing of retrovirus-mediated gene therapy strategies for the treatment of Farber disease. Retroviral vectors were engineered to overexpress AC and a cell surface marker, human CD25. Transduction with these viral vectors corrected the enzymatic defect in Farber patient cells and in vivo administration of the lentiviral vector led to long-term expression of the marking transgene as well as increased AC expression in the liver. To determine the effect of over-expression of AC, human CD34+ cells were transduced and transplanted into NOD/SCID animals. It was found that transgene-expressing cells could reconstitute the host. To address the neurological manifestations of Farber disease, vascular endothelial growth factor (VEGF) was investigated as an agent to transiently open the blood brain barrier for entry of lentivirus. It was found that in addition to increasing the amount of therapeutic virus in the brain, VEGF treatment also increased transduction in other organs. Further, to address the concerns of insertional mutagenesis associated with using integrating vectors, an immunotoxin-based strategy was developed as a safety system to clear transduced cells. It was found that a CD25-targeted immunotoxin could eliminate both transduced hematopoietic cells as well as tumor cells over-expressing CD25. This strategy can be employed following gene therapy should an unwanted proliferative event occur. Together, these studies represent considerable advances towards the development of a cure for Farber disease, demonstrating both therapeutic potential and also containing a built-in safety system.

The p53 protein is a critical tumor suppressor that is mutated in over half of all human cancers. It plays essential roles in maintaining genomic integrity by modulating the cellular response to various types of genotoxic stress. Associating with over 270 proteins to date, one of the mechanisms pivotal to p53’s multifaceted activities is protein-protein interactions. As to how most of these molecules bind to and affect p53 function remains unclear. Here we present a combined structural and biophysical approach to study three p53-interacting partners: BRCA1, IFI16 and p53 affinity reagent in an attempt to elucidate the basis of how these proteins recognize, bind to and alter p53’s biochemical functions. We have biophysically characterized the central region of BRCA1 and examined how it acts as a disordered scaffold to mediate association with p53 and other proteins. Having a putative role as a tumor suppressor, we have determined the crystal structures of the HIN-A and HIN-B domains of IFI16 and find that they interact with the C-terminus and DNA-binding core domain of p53, respectively, and enhance the DNA binding and transactivation activities of p53. Most cancer hot spot mutations of p53 are localized in the core domain and are thermally destabilized. Attaining molecules that stabilize the p53 fold has therefore been regarded as an attractive approach for cancer therapy. Lastly, using a phage-displayed library, evidence is presented to demonstrate a proof-of-principle for generating synthetic affinity reagents to potentially restore the function of tumor-derived p53 core domain mutants.

Single-molecule fluorescence spectroscopy techniques such as Fluorescence Correlation Spectroscopy (FCS) and single-molecule Förster Resonance Energy Transfer (smFRET) not only possess an unprecedented high sensitivity but also have high temporal and spatial resolution. Therefore, they have an immense potential both in investigation of fundamental biological principles and in clinical applications. FCS analyses are based on both theoretical approximations of the beam geometry and assumptions of the underlying molecular processes. To address the accuracy of analysis, firstly the experimental conditions that should be fulfilled in order to obtain reliable physical parameters are discussed and the input parameters are carefully controlled accordingly to demonstrate the performance of FCS measurements on our home-built confocal multiparameter photon-counting microscope in several in vitro and in-vivo applications. Secondly, we performed a comprehensive FCS analysis of rhodamine family of dyes to evaluate the validity of assigning the correlation relaxation times to the time constant of conformational dynamics of biomolecules. While it is the common approach in literature our data suggests that conformational dynamics mainly appear in the correlation curve via modulation of the dark states of the fluorophores. The size and shape of the folded, unfolded and chemically-denatured states of the N-terminal Src-homology-3 of downstream of receptor kinases (DrkN SH3) were investigated by FCS and smFRET burst experiments. Based on the data, we conclude that a considerable sub-population of the denatured protein is in a closed loop state which is most likely formed by cooperative hydrogen bonds, salt bridges and nonpolar contacts. As a clinical application, we developed and characterized an ultrasensitive capillary electrophoresis method on our multiparameter confocal microscope. This allowed us to perform Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with about 500 times better sensivity than a commercial instrument. Quite remarkably, we were able to analyze samples of cell lysate down to the contents of a single cell.

Single particle electron cryomicroscopy (cryo-EM) is a rapidly developing structural biology technique for the study of macromolecular protein complexes. Presently, cryo-EM fills an important niche by facilitating acquisition of 3-D structures of protein complexes not amenable to structure determination by other techniques. Expansion of cryo-EM beyond this niche requires continued improvement in the types of specimens that can be studied as well as the final resolutions achieved. Two studies were undertaken to address these issues. The first examined resolution limitations by quantifying the effect of beam-induced motion in images of beam-sensitive paraffin crystals. The second explored the possibility of using cryo-EM to study the interaction of small effector proteins with a large multi-protein complex, V-ATPase. The results of these studies exposed the fact that fundamental aspects of the imaging and specimen preparation processes remain poorly understood and must be addressed to facilitate future improvements in cryo-EM structure determination.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Jianhan Chen / Intrinsically disordered proteins (IDPs) are functional proteins that lack stable tertiary structures under physiological conditions. IDPs are key components of regulatory networks that dictate various aspects of cellular decision-making, and are over-represented in major disease pathways. For example, about 30% of eukaryotic proteins contain intrinsic disordered regions, and over 70% of cancer-associated proteins have been identified as IDPs. The highly heterogeneous nature of IDPs has presented significant challenge for experimental characterization using NMR, X-ray crystallography, or FRET. These challenges represent a unique opportunity for molecular mod- eling to make critical contributions. In this study, computer simulations at multiple scales were utilized to characterize the structural properties of unbound IDPs as well as to obtain a mechanistic understanding of IDP interactions. These studies of IDPs also reveal significant limitations in the current simulation methodology. In particular, successful simulations of biomolecules not only require accurate molecular models, but also depend on the ability to sufficiently sample the com- plex conformational space. By designing a realistic yet computationally tractable coarse-grained protein model, we demonstrated that the popular temperature replica exchange enhanced sampling is ineffective in driving faster reversible folding transitions for proteins. The second original contribution of this dissertation is the development of novel simulation methods for enhanced sampling of protein conformations, specifically, replica exchange with guided-annealing (RE-GA) method and multiscale enhanced sampling (MSES) method. We expect these methods to be highly useful in generating converged conformational ensembles.

Master of Science / Biochemistry and Molecular Biophysics / Jianhan Chen / Proteins are fundamental building blocks of life in an organism, and to function properly, they must adopt an appropriate three-dimensional conformation or conformational ensemble. In protein aggregation diseases, proteins misfold to incorrect structures that allow them to join together and form aggregates. A wide variety of proteins are involved in these aggregation diseases and there are multiple theories of their disease mechanism. However, a common theme is that they aggregate into filamentous structures. Therapies that target the process by which the aggregating proteins assemble into these similar fibril-like structures may by effective at countering aggregation diseases. This requires models that can accurately describe the assembly process of the fibrils. An analytical theory was recently described where fibrils grow by the templating of peptides onto an existing amyloid core and the kinetics of the templating process is modeled as a random walk in the backbone hydrogen bonding space. In this thesis, I present my work integrating molecular simulation with this analytical model to investigate the dependence of fibril growth kinetics on peptide sequence and other molecular details. Using the Aβ16-22 peptide as a model system, we first calculate the rate matrix of transitions among all possible hydrogen bonding microscopic states using numerous short-time simulations. These rates were then used to construct a kinetic Monte Carlo model for simulations of long-timescale fibril growth. The results demonstrate the feasibility of using such a theory/simulation framework for bridging the significant gap between fibril growth and simulation timescales. At the same time, the study also reveals some limits of describing the fibril growth as a templating process in the backbone hydrogen bonding space alone. In particular, we found that dynamics in nonspecifically bound states must also be considered. Possible solutions to this deficiency are discussed at the end.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / John M. Tomich / Success for gene therapy clinical protocols depends on the design of safe and efficient gene carriers. Nature had already designed efficient DNA or RNA delivery devices, namely virus particles. However, the risk of insertional mutagenesis has limited their clinical use. Alternatively, safer approaches involving non-viral carriers have been and continue to be developed. While they have been reported to be less efficient than viral vectors, adding genome editing elements to pDNA makes the integration of corrective sequence site specific moving non-viral gene delivery systems closer to clinical applications. Over the last decade, peptides have emerged as a new family of potential carriers in gene therapy. Peptides are easy to synthesize, quite stable and expected to produce minimally immunogenic and inflammatory responses. We recently reported on a new class of Branched Amphiphilic Peptides Capsules (BAPCs) that self-assemble into extremely stable nano-spheres. BAPCs display a uniform size of _20 nm if they are incubated at 4_C and they retain their size at elevated temperatures. In the presence of DNA, they can act as cationic nucleation centers around which DNA winds generating peptide-DNA complexes with a size ranging from 50nm to 100nm. However, if BACPs are not incubated at 4_C, the pattern of interaction with DNA differs. Depending of the peptide/DNA ratios, the peptides either coat the plasmid surface forming nano-_bers (0.5-1 _M in length) or condense the plasmid into nano-sized structures (100-400nm). Different gene delivery efficiencies are observed for the three types of assemblies. The structure where the DNA wraps around BAPCs display much higher transfection efficiencies in HeLa cells in comparison to the other two morphologies and the commercial lipid reagent Lipofectinr. As a proof of concept, pDNA was delivered in vivo, as a vaccine DNA encoding E7 oncoprotein of HPV-16. It elicited an immune response activating CD8+ T cells and provided anti-tumor protection in a murine model.

Doctor of Philosophy / Biochemistry and Molecular Biophysics / Om Prakash / John M. Tomich / As part of the ongoing development of a channel forming peptide with the potential to be used clinically to treat cystic fibrosis, a number of structural studies using solution NMR spectroscopy have been carried out on a number of the test sequences. Given their structural similarities of the monomers it is important to evaluate whether or not there is a compelling need to determine the solution NMR structure of next-generation peptides. The determination of the NMR monomeric solution structure of peptides NK₄-M2GlyR-p22 and NK₄-M2GlyR-p20 T17R S20W in TFE solution and SDS micelles sample shows predominantly alpha-helical conformations for both sequences with an extended conformation for the N-terminal lysine residues. The I[subscript max], K[subscript 1/2] and Hill coefficient, derived from data on ion conductance across monolayers of MDCK cells, were used to compare the ion conductance properties of the peptide sequences. Peptide NK₄ M2GlyR p20 T17R S20W has both a higher I[subscript MAX] (43.8 ± 2.8 μA/cm²) and a lower K[subscript 1/2] (58 ± 8 μM) compared to other M2GlyR derived peptides with calculated NMR structures. All available molecular structures calculated by NMR for M2GlyR derived peptides were compared and the correlation of the structural changes observed in the NMR structures with the ion conductance changes was evaluated. The NMR structures were found to have limited predicting potential over the ion conduction data. NMR determined structures have provided an experimentally based starting point for studies of the channels formed by the family of M2GlyR peptides. Computer simulations account for inter peptide interactions and packing effects that are not experienced by the monomeric form of the peptides in the NMR samples that have been used until now. The determination of the structure of the oligomeric peptide channels is deemed needed to improve the relevance of future use of NMR in this project. The use of larger membrane mimicking agents, isotopically labeled (¹⁵N, ¹³C) samples, 3D NMR experiments and potentially solid state NMR would be required to accomplish that task.