Master of Science / Biochemistry and Molecular Biophysics / John M. Tomich / NC-1007 (CK₄-M2GlyR) (PARVGLGITTVLTMTTQSSGSRAKKKK) is a synthetic peptide modeled after the second transmembrane segment of the spinal cord glycine receptor’s α-subunit, and has demonstrates the capacity to oligomerize to form transmembrane channels with Cl[superscript]- permselectivity. While studies into the effects of truncation on both CK[subcript]4 (C-terminal tetra-lysl adducted) and NK[subscript]4 (N-terminal tetra-lysl adducted) led to more control over solution aggregation in the NK[subscript]4 variant, the work presented explore whether C-terminal sequential substitutions with a tryptophan residue could similarly stabilize the aqueous structure in monomeric form or further define the pore registry in such a way as to promote an increase ion permeability. Tryptophan was substituted for amino acids in the 18[superscript]th, 19[superscript]th, 20[superscript]th, and 21[superscript]st positions of the peptide sequence (SSGS, respectively), and changes in aggregation profiles, secondary structure, and channel ion permeability were observed. Synthesized peptides show circular dichroism spectral profiles indicating that the studied tryptophan substitutions did not result in a reduction of the characteristic helicity of the peptide; however, the tryptophan substitution also did little to decrease solution aggregation as demonstrated by comparative studies by reverse-phase high- performance liquid chromatography. All peptides demonstrated channel activity, directly measured by recordings of transepithelial short-circuit current. with profiles that suggest trends in electrostatic interactions and membrane registry relative to substitution position. One peptide in particular, NC-1007 S21W displayed atypical activity, which could not be effectively described by the standard Hill-based model but may be indicative of an ill-defined registry due to the substituted peptide’s proximity to another strongly pore-defining residue. Further studies in the effects of sequence modification to channel-forming peptides will elucidate how sequences may be altered to optimize synthetic peptide solubility, resistance to in-solution aggregation, and ability to form selective and permeable ion channels. The understanding gained from this study will improve our ability to develop peptides that could serve as a therapeutic treatments for a number of endogenous channelopathies.

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.

Doctor of Philosophy / Department of Chemistry / Ryszard J. Jankowiak / The constituents of oxygen-evolving photosystem II core complexes—antenna proteins (CP43 and CP47) and reaction center (RC)—have been the subject of many studies over the years. However, the various issues related to electronic structure, including the origin/composition of the lowest-energy traps, origin of various emission bands, excitation energy transfer (EET), primary charge separation (CS) processes and pigment site energies remain yet to be fully resolved. Exploiting our state-of-the-art techniques such as low-T absorption, fluorescence, and hole burning (HB) spectroscopies, we resolved some of the issues particularly related to CP47 and isolated RC protein complexes. For example, we demonstrated that the fluorescence origin band maximum (~695 nm) originates from the lowest-energy state ~693 nm of intact CP47. In intact CP47 in contrast to destablished protein complexes, the band (~695 nm) does not shift in the temperature range of 5–77 K unless hole-burning takes place. We also studied a large number of isolated RC preparations from spinach, and wild-type Chlamydomonas reinhardtii (at different levels of intactness), as well as its mutant (D2-L209H), in which the active branch pheophytin (PheoD1) has been genetically replaced with chlorophyll a (Chl a). We showed that the Qx-/Qy-region site-energies of PheoD1 and PheoD2 are ~545/680 nm and ~541.5/670 nm, respectively, in good agreement with our previous assignment [Jankowiak et al. J. Phys. Chem. B 2002, 106, 8803]. Finally, we demonstrated that the primary electron donor in isolated algal RCs from C. reinhardtii (referred to as RC684) is PD1 and/or PD2 of the special Chl pair (analogous to PL and PM, the special BChl pair of the bacterial RC) and not ChlD1. However, the latter can also be the primary electron donor (minor pathway) in RC684 depending on the realization of the energetic disorder. We further demonstrate that transient HB spectra in RC684 are very similar to P+QA - PQA spectra measured in PSII core, providing the first evidence that RC684 represent intact isolated RC that also possesses the secondary electron acceptor, QA. In summary, a new insight into possible charge separation pathways in isolated PSII RCs has been provided.

Doctor of Philosophy / Department of Biochemistry / Ramaswamy Krishnamoorthi / Insect [beta]-1,3-glucan recognition protein ([beta]GRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Delineation of mechanistic details of these processes may help develop strategies to control insect-borne diseases and economic losses. Multi-dimensional nuclear magnetic resonance (NMR) techniques were employed to solve the solution structure of the Indian meal moth (Plodia interpunctella) [beta]GRP N-terminal domain (N-[beta]GRP), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. This is the first determined three-dimensional structure of N-[beta]GRP, which adopts an immunoglobulin fold. Addition of laminarin, a [beta]-1,3 and [beta]-1,6 link-containing glucose polysaccharide (∼6 kDa) that activates the proPO pathway, to N-[beta]GRP results in the loss of NMR cross-peaks from the backbone [subscript]1[subscript]5N-[subscript]1H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-[beta]GRP:laminarin complex show that ligand binding induces self-association of the protein-carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (∼102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to submicromolar concentrations. The structural model thus derived from this study for the N-[beta]GRP:laminarin complex in solution differs from the one in which a single N-[beta]GRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of a X-ray crystal structure of the N-[beta]GRP:laminarihexaose complex. AUC studies and phenoloxidase activation measurements made with designed mutants of N-[beta]GRP indicate that electrostatic interactions between the ligand-bound protein molecules contribute to the stability of the N-[beta]GRP:laminarin complex and that a decreased stability results in a reduction of proPO activation. These novel findings suggest that ligand-induced self-association of the [beta]GRP:[beta]-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the pathogen recognition signal. In the case of the homolog of GNBPA2 from Anopheles gambiae, the malaria-causing Plasmodium carrier, multiligand specificity was characterized, suggesting a functional diversity of the immunoglobulin domain structure.

Doctor of Philosophy / Biochemistry and Molecular Biophysics / Phillip E. Klebba / The goal of the research included in this dissertation is to provide a more complete model of the role of TonB, an energy transducing protein that resides in the inner membrane and is an essential component of the iron transport of Escherichia coli under iron-starved conditions. Using fluorescent hybrid proteins, the anisotropy of TonB in the cytoplasmic membrane (CM) of Escherichia coli was determined. With the aim of understanding the bioenergetics of outer membrane (OM) iron transport, the dependence of TonB motion on the electrochemical gradient and the effect of CM proteins ExbB and ExbD on this phenomenon was monitored and analyzed. The native E. coli siderophore, enterobactin chelates Fe⁺³ in the environment and ferric enterobactin (FeEnt) enters the cell by energy- and TonB-dependent uptake through FepA, its OM transporter. The TonB-ExbBD complex in the CM is hypothesized to transfer energy to OM transporters such as FepA. We observed the polarization of GFPTonB hybrid proteins and used metabolic inhibitors (CCCP, azide and dinitrophenol) and chromosomal deletions of exbBD to study these questions. The results showed higher anisotropy (R) values for GFP-TonB in energy-depleted cells, and lower R-values in bacteria lacking ExbBD. Metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings suggest that TonB undergoes constant, energized motion in the bacterial CM, and that ExbBD mediates its coupling to the electrochemical gradient. By spectroscopic analyses of extrinsic fluorophore labeled site-directed Cys residues in 7 surface loops of Escherichia coli FepA, binding and transport of ferric enterobactin (FeEnt) was characterized. Changes in fluorescence emissions reflected conformational motion of loops that altered the environment of the fluorophore, and we observed these dynamics as quenching phenomena during FeEnt binding and transport in living cells or outer membrane vesicles. Cys residues in each of the 7 surface loops (L2, L3, L4, L5, L7 L8, and L11) behaved individually and characteristically with regard to both fluorophore maleimide reactivity and conformational motion. Fluorescence measurements of FeEnt transport, by either microscopic or spectroscopic methodologies, demonstrated that ligand uptake occurs uniformly throughout the cell envelope, and susceptibility of FeEnt uptake to the proton ionophore m-chlorophenyl hydrazone (CCCP) at concentrations as low as 5 uM. The latter result recapitulates the sensitivity of inner membrane major facilitator transporters to CCCP (Kaback, 1974), providing further evidence of the electrochemical gradient as a driving force for TonB-dependent metal transport.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / John M. Tomich / Branched Amphiphilic Peptide Capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble in water to form bilayer delimited poly-cationic capsules capable of trapping solutes. We examined the lipid-like properties of this system including assembly, fusion, solute encapsulation, and resizing by membrane extrusion as well as their capability to be maintained at a specific size by storage at 4˚C. These studies along with earlier work from the lab (Gudlur et al. (2012) PLOS ONE 7(9): e45374) demonstrated that the capsules, while sharing many properties with lipid vesicles, were much more robust. We next investigated the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs. We demonstrated that the BAPCs are readily taken up by epithelial cells in culture, escape or evade the endocytotic pathway, and accumulate in the peri-nuclear region where they persist without any apparent degradation. The stability and persistence of the capsules suggested they might be useful in delivering radionuclides. The BAPCs encapsulated alpha particle emitting radionuclides without any apparent leakage, were taken up by cells and were retained for extended periods of time. Their potential in this clinical application is being currently pursued. Lastly we studied the temperature dependence of capsule formation by examining the biophysical characteristics of temperature induced conformational changes in BAPCs and examined the structural parameters within the sequences that contribute to their remarkable stability. A region in the nine-residue sequence was identified as the critical element in this process. The ability to prepare stable uniform nano-scale capsules of desired sizes makes BAPCs potentially attractive as delivery vehicles for various solutes/drugs.

Master of Science / Department of Physics / Robert Szoszkiewicz / Proteins are involved in many of the essential cellular processes, such as cell adhesion, muscle function, enzymatic activity or signaling. It has been observed that the biological function of many proteins is critically connected to their folded conformation. Thus, the studies of the process of protein folding have become one of the central questions at the intersection of biophysics and biochemistry. We propose to use the changes of the nanomechanical properties of these biomolecules as a proxy to study how the single proteins fold. In the first steps towards this goal, the work presented in this thesis is concentrated on studies of unfolding forces and pathways of one particular multidomain protein, as well as on development of the novel method to study elastic spring constant and mechanical energy dissipation factors of simple proteins and peptides. In the first part of this thesis we present the results of the mean unfolding forces of the NRR region of the Notch1 protein. Those results are obtained using force spectroscopy techniques with the atomic force microscope (AFM) on a single molecule level. We study force-induced protein unfolding patterns and relate those to the conformational transitions within the protein using available crystal structure of the Notch protein and molecular dynamics simulations. Notch is an important protein, involved in triggering leukemia and breast cancers in metazoans, i.e., animals and humans. In the second part of this thesis we develop a model to obtain quantitative measurements of the molecular stiffness and mechanical energy dissipation factors for selected simple proteins and polypeptides from the AFM force spectroscopy measurements. We have developed this model by measuring the shifts of several thermally excited resonance frequencies of atomic force microscopy cantilevers in contact with the biomolecules. Next, we provided partial experimental validation of this model using peptide films. Ultimately, our results are expected to contribute in the future to the developments of medical sciences, which are advancing at a level, where human health and disease can be traced down to molecular scale.