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.

Doctor of Philosophy / Department of Chemistry / Paul E. Smith / In the study of biological systems, molecular dynamics (MD) simulations have played an important role in providing atomic details for phenomena of interest. The force field used in MD simulations is a critical factor determining the quality of the simulations. Recently, Kirkwood-Buff (KB) theory has been applied to study preferential interactions and to develop a new force field. KB theory provides a path from quantities determined from simulation data to the corresponding thermodynamic data. Here we combine KB theory and molecular simulations to study a variety of intermolecular interactions in solution. First, recent results concerning the formulation and evaluation of preferential interactions in biological systems in terms of KB integrals are presented. In particular, experimental and simulated preferential interactions of a cosolvent with a biomolecule in the presence of water are described. Second, a force field for the computer simulation of aqueous solutions of amides is presented. The force field is designed to reproduce the experimentally observed density and KB integrals for N-methylacetamide (NMA), allowing for an accurate description of the NMA activity. Other properties such as the translational diffusion constant and heat of mixing are also well reproduced. The force field is then extended to include N,N'-dimethylacetamide and acetamide with good success. The models presented here provide a basis for an accurate force field for peptides and proteins. Comparison between the developed KB force fields (KBFF) and existing force fields is performed for amide and glycine and proves that the KBFF approach is competitive. Also, explicit expressions are developed for the chemical potential derivatives, partial molar volumes, and isothermal compressibility of solution mixtures involving four components at finite concentrations using the KB theory of solutions. A general recursion relationship is also provided which can be used to generate the chemical potential derivatives for higher component solutions. Finally, a pairwise preferential interaction model (PPIM), described by KB integrals is developed to quantify and characterize the interactions between functional groups observed in peptides.

Master of Science / Department of Biochemistry / John M. Tomich / Biologically based adhesives offer many industrial advantages over their chemically synthesized counterparts, not the least of which are reduced environmental impact and limited toxicity. They also represent a renewable resource. In addition, nanoscale biomaterials also show an incredibly large potential for biomedical uses, including possible drug delivery and novel wound bandaging, as well as tissue engineering. Understanding the adhesion mechanisms at work in peptide-based nanomaterials is key for producing viable industrial and clinical biomimetic compounds. Our previous work has shown that small hydrophobic oligopeptide segments flanked by short tri-lysine sequences display adhesion strength that is dependent on the formation of β-structure and large-scale association of monomers. In this study, three oligopeptides were synthesized based on putative amyloid fibril nucleation sites. Two of the sequences originate from the Alzheimer’s beta amyloid peptide Aβ1-40, while the third sequence comes from a nucleation site for islet amyloid polypeptide (IAPP). These peptides show unusual structural properties associated with adhesive ability. Furthermore, they represent a third category of requirements for β-structure formation. In addition, I report the first morphological evidence for the previously predicted structural mechanism underlying our previous peptide based adhesives.

Motivé par l’évolution de la production architecturale durable dans les pays d’Amérique latine, et plus particulièrement en Colombie, mon projet de recherche porte sur l’adaptation de l’architecture à ce nouveau contexte. L’approche architecturale traditionnelle à la prise en compte de l’énergie et du climat est l’architecture bioclimatique : reproduite à partir de connaissances et techniques ancestrales remontant à la conception de l’abri, cette dernière étudie les phénomènes physiques associés au confort thermique afin de les reproduire dans une nouvelle architecture. De nouvelles méthodes d’évaluation environnementale se sont développées dans les dernières décennies pour améliorer l’intégration environnementale des bâtiments. Ces méthodes privilégient la normalisation des solutions et utilisent des systèmes de certification pour reconnaître la performance environnementale et énergétique des bâtiments. Le résultat visé est la conformité aux standards internationaux de durabilité. Ce mémoire porte sur l’analyse comparative de l’architecture bioclimatique et de la certification environnementale à partir de la structure des sujets abordés par LEED, une des méthodes les plus connues d’une telle certification. Cette comparaison permet de constater que les deux approches sont motivées par les mêmes préoccupations environnementales mais que leurs méthodes d’intégration de ces préoccupations diffèrent, en particulier quant à la prise en compte des facteurs locaux et globaux. / Motivated by the development of sustainable architectural production in the countries of Latin America, particularly in Colombia, my research focuses on the adaptation of architecture to this new context. The traditional approach to the integration of energy and climate is bioclimatic architecture. This way is based on the reproduction of knowledge through the use of ancestral techniques that were acquired over time, and which evolved from shelter design. The bioclimatic architecture studies the physical phenomena in relation to thermal comfort to reproduce in a new architectural style. New environmental assessment methods have been developed in recent decades to improve environmental integration in buildings. These methods give priority to standardization of solutions and use certification systems to recognize the environmental and energy performance of buildings. The expected results are in compliance with international sustainability standards. This thesis focuses on the comparative analysis of bioclimatic architecture and environmental certification using the structure and the topics addressed by LEED, one of the best-known methods of such certification. This comparison shows that the two approaches are motivated by the same environmental concerns but their methods of integration of these concerns differ, particularly with regard to the consideration of local and global factors.

AcrA is the periplasmic component of an efflux system AcrA-AcrB-TolC, which can expel different classes of antibiotics. AcrB is the inner membrane (IM) pump that utilizes proton-motive force for the active transport, TolC is the outer membrane (OM) channel, and AcrA coordinates the actions of AcrB and TolC, so that substrates are expelled across the two membranes, bypassing the periplasm. It has been proposed that AcrA either provides a static seamless link between AcrB and TolC, or acts like its analogous viral membrane fusion protein (MFP) and actively brings the IM and OM closer for substrate transfer. To better understand the role of AcrA in the efflux mechanism, site-directed spin labeling (SDSL)/EPR (electron paramagnetic resonance) spectroscopy is used to investigate the structure and dynamics of AcrA in solution. My results demonstrated that AcrA is a dynamic protein that undergoes pH-dependent and reversible conformational changes. AcrA contains an interrupted alpha-helical, coiled-coil domain flanked by a pair of beta-stranded lipoyl motifs, and my SDSL/EPR analysis revealed that the pH-induced conformation change mainly involves the coiled-coil and the lipoyl domains. In addition, I found that each AcrA monomer folds into an intra-molecular hairpin and AcrA monomers oligomerize with their coiled-coil hairpins aligned in parallel. Unlike the pH-induced conformational rearrangement of a viral MFP, change in pH alters both intra- and inter-molecular interaction along the coiled-coil of AcrA without rearranging the hairpin fold. The organization of AcrA protomers and its pH-induced conformational switching are, however, congruent with the TolC coiled-coil hairpins in the iris-like opening of the TolC channel. Together, my studies suggest that rather than being a passive structural linkage between AcrB and TolC, AcrA plays an active role mediating the drug efflux. The reported AcrA dynamics provides new insights into the AcrA-TolC interactions for the channel opening during the efflux process.

Single particle electron cryomicroscopy (cryo-EM) is a method of structure determination that uses many randomly oriented images of the specimen to construct a three-dimensional density map. In this thesis, single particle cryo-EM has been used to determine the structure of intact adenosine triphosphate (ATP) synthase from bovine heart mitochondria, an approximately 550 kDa membrane protein complex. In respiring organisms, ATP synthase is responsible for synthesizing the majority of ATP, a molecule that serves as an energy source for many cellular reactions. In order to understand the mechanism of ATP synthase, knowledge of the arrangement of subunits in the intact complex is necessary. To obtain maps of intact ATP synthase showing internal density distributions by single particle cryo-EM, methodological improvements to image acquisition, map refinement, and data selection were developed. Further, a novel segmentation algorithm was developed to aid in interpretation of maps. The use of these tools allowed for construction and interpretation of two maps of ATP synthase, solubilized in different membrane mimetics, in which the arrangement of subunits could be identified. These maps revealed interactions within the complex important for its function. In addition, evidence was obtained for curvature of membrane mimetics around ATP synthase, suggesting a role for the complex in maintenance of mitochondrial membrane morphology.

Single particle electron cryomicroscopy (cryo-EM) is a method of structure determination that uses many randomly oriented images of the specimen to construct a three-dimensional density map. In this thesis, single particle cryo-EM has been used to determine the structure of intact adenosine triphosphate (ATP) synthase from bovine heart mitochondria, an approximately 550 kDa membrane protein complex. In respiring organisms, ATP synthase is responsible for synthesizing the majority of ATP, a molecule that serves as an energy source for many cellular reactions. In order to understand the mechanism of ATP synthase, knowledge of the arrangement of subunits in the intact complex is necessary. To obtain maps of intact ATP synthase showing internal density distributions by single particle cryo-EM, methodological improvements to image acquisition, map refinement, and data selection were developed. Further, a novel segmentation algorithm was developed to aid in interpretation of maps. The use of these tools allowed for construction and interpretation of two maps of ATP synthase, solubilized in different membrane mimetics, in which the arrangement of subunits could be identified. These maps revealed interactions within the complex important for its function. In addition, evidence was obtained for curvature of membrane mimetics around ATP synthase, suggesting a role for the complex in maintenance of mitochondrial membrane morphology.

The Escherichia coli acid stress response allows the survival of cells over a wide range of pH challenges: down to pH 2.0 with the extreme acid stress response and down to pH 4.0 – 5.0 with the mild acid stress response. The cell employs a number of different acid stress response systems, including a number of structurally related, pyridoxal-5′-phosphate (PLP)-dependent amino acid decarboxylases, including the glutamic acid, arginine, lysine, and ornithine decarboxylases. The decarboxylases are large multi-domain enzymes that exist as homodimers or higher-order oligomers and have various activity optima at different pH values. By the proton-consuming decarboxylation of a target amino acid, these enzymes provide a response to a wide range of pH challenges. The primary focus of this work is the elucidation of the X-ray crystal structure of the inducible lysine decarboxylase LdcI, a homodecameric enzyme that has distinct 5-fold symmetry. A combination of heavy-atom derivatization, anomalous scattering and molecular replacement techniques were used to determine the X-ray structure and the model was refined to a resolution of 2.0 Å. The structure of LdcI revealed that the protein co-crystallized with the stringent response alarmone ppGpp. The stringent response is activated under nutritional and stress conditions and reorganizes cellular transcription and metabolism from exponential-phase growth into stationary phase growth. The primary target of ppGpp is the RNA polymerase, but other classes of enzymes are known to be affected. ppGpp was found to be a potent inhibitor of LdcI both in vitro and in vivo and this role provides the first evidence of a linkage between the stringent response and acid stress response. Among the decarboxylases related to LdcI (the constitutive lysine, the ornithine and arginine decarboxylases), a number of these enzymes were similarly regulated by ppGpp.

AcrA is the periplasmic component of an efflux system AcrA-AcrB-TolC, which can expel different classes of antibiotics. AcrB is the inner membrane (IM) pump that utilizes proton-motive force for the active transport, TolC is the outer membrane (OM) channel, and AcrA coordinates the actions of AcrB and TolC, so that substrates are expelled across the two membranes, bypassing the periplasm. It has been proposed that AcrA either provides a static seamless link between AcrB and TolC, or acts like its analogous viral membrane fusion protein (MFP) and actively brings the IM and OM closer for substrate transfer. To better understand the role of AcrA in the efflux mechanism, site-directed spin labeling (SDSL)/EPR (electron paramagnetic resonance) spectroscopy is used to investigate the structure and dynamics of AcrA in solution. My results demonstrated that AcrA is a dynamic protein that undergoes pH-dependent and reversible conformational changes. AcrA contains an interrupted alpha-helical, coiled-coil domain flanked by a pair of beta-stranded lipoyl motifs, and my SDSL/EPR analysis revealed that the pH-induced conformation change mainly involves the coiled-coil and the lipoyl domains. In addition, I found that each AcrA monomer folds into an intra-molecular hairpin and AcrA monomers oligomerize with their coiled-coil hairpins aligned in parallel. Unlike the pH-induced conformational rearrangement of a viral MFP, change in pH alters both intra- and inter-molecular interaction along the coiled-coil of AcrA without rearranging the hairpin fold. The organization of AcrA protomers and its pH-induced conformational switching are, however, congruent with the TolC coiled-coil hairpins in the iris-like opening of the TolC channel. Together, my studies suggest that rather than being a passive structural linkage between AcrB and TolC, AcrA plays an active role mediating the drug efflux. The reported AcrA dynamics provides new insights into the AcrA-TolC interactions for the channel opening during the efflux process.

A combined attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR)-atomic force microscopy (AFM) platform was used to visualize and characterize membranolytic protein- and peptide-membrane interactions, allowing spectroscopic details to be correlated with structural features. Modifications to a previous combined platform permitted IR results for physiologically-relevant protein or peptide concentrations as well as provided nanometer-resolution height data for AFM. This combination provides greater insight than individual techniques alone. The interactions of hemolytic sticholysin proteins on a model red blood cell membrane showed evidence of conformational changes associated with a membrane-induced organization. In addition, the examination of a de novo cationic antimicrobial peptide on a model bacterial membrane showed that the peptide adopted a helical structure upon interaction with the membrane, and also provided evidence of membrane disruption and peptide aggregation. These results demonstrate that ATR-FTIR-AFM can be a powerful tool for understanding protein- and peptide-membrane interactions.