Biological interactions occur on multiple length scales, ranging from molecular to population wide interactions. This work describes the study of two specific areas of biological interactions in microbial systems: intracellular protein-protein interactions and cell-to-cell interactions. The implementation of optical and atomic force microscopy and the methodologies developed during this study proved to be invaluable tools for investigating these systems. Identifying and characterizing protein interactions are fundamental steps toward understanding complex cellular networks. We have developed a unique methodology which combines an imaging-based protein interaction assay with a fluorescence recovery after photobleaching technique (FRAP). Protein interactions are readily detected by co-localization of two proteins of interest fused to green fluorescent protein (GFP) and DivIVA, a cell division protein from Bacillus subtilis. We demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions over four orders of magnitude. FRAP data was analyzed using a combination of various image processing techniques and analytical models. This combined approach made it possible to estimate cell morphology parameters such as length, diameter, the effective laser probe volume, as well as to the mobile protein concentration in vivo, the number of bound molecules at the cellular poles, and the biophysical parameter koff. Cells not only utilize molecular interactions in the intracellular environment, but also express proteins, polysaccharides and other complex molecules to mediate interactions with the surrounding extracellular environment. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting cell-to-cell interactions. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Further analyses suggest that the extracellular matrix differs between the cheA1 and the cheY1 deletion mutants, despite similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition.

Calcium regulates many biological processes through interaction with proteins with different conformational, dynamic, and metal binding properties. Previous studies have shown that the electrostatic environment plays a key role in calcium binding affinity. In this research, we aim to dissect the contribution of the electrostatic environment to calcium binding affinity using protein design. Many natural calcium binding proteins undergo large conformational changes upon calcium binding which hampers the study of these proteins. In addition, cooperativity between multiple calcium binding sites makes it difficult to study site-specific binding affinity. The design of a single calcium binding site into a host system eliminates the difficulties that occur in the study of calcium binding affinity. Using a computer algorithm we have rationally designed several calcium binding sites with a pentagonal bipyramidal geometry in the non-calcium dependent cell adhesion protein CD2 (CD2-D1) to better investigate the key factors that affect calcium binding affinity. The first generation proteins are all in varying electrostatic environments. The conformational and metal binding properties of each of these designed proteins were analyzed. The second generation designed protein, CD2.6D79, was designed based on criteria learned from the first generation proteins. This protein contains a novel calcium binding site with ligands all from the â-strands of the non-calcium dependent cell adhesion protein CD2. The resulting protein maintains native secondary and tertiary packing and folding properties. In addition to its selectivity for calcium over other mono and divalent metal ions, it displays strong metal binding affinities for calcium and its analogues terbium and lanthanum. Furthermore, our designed protein binds CD48, the ligand binding partner of CD2, with an affinity three-fold stronger than CD2. The electrostatic potential of the calcium binding site was modified through mutation to facilitate the study of the effect of electrostatic interactions on calcium binding affinity. Several charge distribution mutants display varying metal binding affinities based on their charge, distance to the calcium binding site, and protein stability. This study will provide insight into the key site factors that control calcium binding affinity and calcium dependent biological function.

RNA aptamers are in vitro sequences of RNA that have a high affinity for their target ligand. They have applications in therapeutics, biosensors and molecular machines. While the practical applications of aptamers are increasing, it is important to study their structure and thermodynamics to improve the understanding of these molecular tools. The malachite green aptamer (MGA) provides a model system to study the interactions between aptamer and ligand that do not involve hydrogen bonding between ligand and receptor. While the original application of this aptamer was abandoned, study of the MGA binding pocket revealed an electronegative environment that was harnessed for catalysis. MGA binding also supported the notion that aptamers bind by adaptive binding. Adaptive binding is the ability of molecules to mold themselves around the structure of a ligand thereby incorporating it into their three-dimensional fold. To further expand our understanding of MGA binding and to clarify conflicting reports of affinities, we conducted isothermal calorimetry binding studies. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. This data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands, whereas, the complex with the original selection target is stable at low salt and in the absence of divalent metal ions. Next, competitive binding studies using isothermal titration calorimetry were conducted with the aim of understanding the adaptive nature of RNA. The results of these studies reveal that there are limits to the adaptability of the aptamer. Binding of one type of ligand reduces the affinity of the aptamer pocket to a differently shaped ligand, even if this second ligand has a significantly higher affinity. The ability of MGA to change ligand preference based on buffer conditions, and the previously reported catalysis suggested that RNA may have a potential supporting multiple functions in the same molecule. To investigate this possibility we attempted to select an aptamer that supports both ligand binding and catalysis. By conducting both a DNA and RNA selection we hoped to add to the iv collection of DNA and RNA aptamers selected for the same target. There are currently too few of these to determine if any correlation can be made between DNA and RNA sequences that bind the same target. The target of the selection was fluorescein diacetate (FDA), which was chosen with the aim that it would allow the exploration of the inherent potential of the selected aptamer to cleave FDA to fluorescein. The RNA selection proved to be more successful and an attempt was made to characterize the binding of the aptamer to its target fluorescein diacetate. Unfortunately there were complications with the binding assays, but future work is proposed that should address the issues. In order to expand the MGA catalytic repertoire attempts were made to synthesize new ligands that could exploit the catalytic potential of the MGA binding pocket. Unfortunately these attempts were unsuccessful, however further attempts are recommended. The MGA used in this study was transcribed in vitro using T7 RNA polymerase. This process is known to add extra nucleotides to the end of the transcription product. Attempts were made to eliminate the n+1 product by introducing a ribozyme or DNAzyme. These were met with difficulties resulting in low yield, however mass spectrometry revealed that n and n+1 MGA bind to ligand. This, along with secondary structure prediction suggests that MGA n+1 behaves the same as n. Overall, the results presented here provide insights into the capabilities of RNA aptamers with respect to ligand binding and catalysis.

No description available.

RNA aptamers are in vitro sequences of RNA that have a high affinity for their target ligand. They have applications in therapeutics, biosensors and molecular machines. While the practical applications of aptamers are increasing, it is important to study their structure and thermodynamics to improve the understanding of these molecular tools. The malachite green aptamer (MGA) provides a model system to study the interactions between aptamer and ligand that do not involve hydrogen bonding between ligand and receptor. While the original application of this aptamer was abandoned, study of the MGA binding pocket revealed an electronegative environment that was harnessed for catalysis. MGA binding also supported the notion that aptamers bind by adaptive binding. Adaptive binding is the ability of molecules to mold themselves around the structure of a ligand thereby incorporating it into their three-dimensional fold. To further expand our understanding of MGA binding and to clarify conflicting reports of affinities, we conducted isothermal calorimetry binding studies. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. This data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands, whereas, the complex with the original selection target is stable at low salt and in the absence of divalent metal ions. Next, competitive binding studies using isothermal titration calorimetry were conducted with the aim of understanding the adaptive nature of RNA. The results of these studies reveal that there are limits to the adaptability of the aptamer. Binding of one type of ligand reduces the affinity of the aptamer pocket to a differently shaped ligand, even if this second ligand has a significantly higher affinity. The ability of MGA to change ligand preference based on buffer conditions, and the previously reported catalysis suggested that RNA may have a potential supporting multiple functions in the same molecule. To investigate this possibility we attempted to select an aptamer that supports both ligand binding and catalysis. By conducting both a DNA and RNA selection we hoped to add to the iv collection of DNA and RNA aptamers selected for the same target. There are currently too few of these to determine if any correlation can be made between DNA and RNA sequences that bind the same target. The target of the selection was fluorescein diacetate (FDA), which was chosen with the aim that it would allow the exploration of the inherent potential of the selected aptamer to cleave FDA to fluorescein. The RNA selection proved to be more successful and an attempt was made to characterize the binding of the aptamer to its target fluorescein diacetate. Unfortunately there were complications with the binding assays, but future work is proposed that should address the issues. In order to expand the MGA catalytic repertoire attempts were made to synthesize new ligands that could exploit the catalytic potential of the MGA binding pocket. Unfortunately these attempts were unsuccessful, however further attempts are recommended. The MGA used in this study was transcribed in vitro using T7 RNA polymerase. This process is known to add extra nucleotides to the end of the transcription product. Attempts were made to eliminate the n+1 product by introducing a ribozyme or DNAzyme. These were met with difficulties resulting in low yield, however mass spectrometry revealed that n and n+1 MGA bind to ligand. This, along with secondary structure prediction suggests that MGA n+1 behaves the same as n. Overall, the results presented here provide insights into the capabilities of RNA aptamers with respect to ligand binding and catalysis.

Changes in spontaneous spike activities recorded from murine frontal cortex networks grown on substrate-integrated microelectrodes were used to determine the dissociation constant (KB) of three GABAA antagonists. Neuronal networks were treated with fixed concentrations of GABAA antagonists and titrated with muscimol, a GABAA receptor agonist. Muscimol decreased spike activity in a concentration dependent manner with full efficacy (100% spike inhibition) and a 50% inhibitory concentration (IC50) of 0.14 ± 0.05 µM (mean ± SD, n=6). At 10, 20, 40 and 80 µM bicuculline, the muscimol IC50 values were shifted to 4.3 ± 1.8 µM (n=6), 6.8 ± 1.7 µM (n=6), 19.3 ± 3.54 µM (n=10) and 43.5 µM (n=2), respectively (mean ± SD). Muscimol titration in the presence of 10, 20, 40 µM of gabazine resulted in IC50s values of 20.1 (n=2), 37.17 (n=4), and 120.45 (n=2), respectively. In the presence of 20, 80, and 160 µM of TMPP (trimethylolpropane phosphate) the IC50s were 0.86 (n=2), 3.07 (n=3), 6.67 (n=2) µM, respectively. Increasing concentrations of GABAA antagonists shifted agonist log concentration-response curves to the right with identical efficacies, indicating direct competition for the GABAA receptor. A Schild plot analysis with linear regression resulted in slopes of 1.18 ± 0.18, 1.29 ± 0.23 and 1.05 ± 0.03 for bicuculline, gabazine and TMPP, respectively. The potency of antagonists was determined in terms of pA2 values. The pA2 values were 6.63 (gabazine), 6.21 (bicuculline), and 5.4 (TMPP). This suggests that gabazine has a higher binding affinity to the GABAA receptor than bicuculline and TMPP. Hence, using spike rate data obtained from population responses of spontaneously active neuronal networks, it is possible to determine key pharmacological properties of drug-receptor interactions.

Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N-acetyl-2-aminofluorene-, 1-amino-pyrene-, BPDE (benzo[a]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking bulky adduct, (+)-cis-anti-BPDE-dG, we demonstrated that, on binding of RPA. the fluorescence of BPDE-ssDNA was significantly enhanced by up to 8-9-fold. This indicated that the stacking between the BPDE adduct and its neighbouring ssDNA bases had been disrupted and there was a lack of substantial direct contacts between the protein residues and the lesion itself. For RPA interaction with short damaged ssDNA, we propose that, on RPA binding, the modified base of ssDNA is looped out from the surface of the protein, permitting proper contacts of RPA with the remaining unmodified bases.

京都大学 / 0048 / 新制・課程博士 / 博士(ｴﾈﾙｷﾞｰ科学) / 甲第20775号 / エネ博第360号 / 新制||エネ||71(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 木下 正弘 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

Yes / The use of peptides to nucleate, grow, and stabilize nanoparticles in aqueous media via non-covalent interactions offers new possibilities for creating functional, water-dispersed inorganic/organic hybrid materials, particularly for Au nanoparticles. Numerous previous studies have identified peptide sequences that both possess a strong binding affinity for Au surfaces and are capable of supporting nanoparticle growth in water. However, recent studies have shown that not all such peptide sequences can produce stable dispersions of these nanoparticles. Here, via integrated experiments and molecular modeling, we provide new insights into the many factors that influence Au nanoparticle growth and stabilization in aqueous media. We define colloidal stability by the absence of visible precipitation after at least 24 hours post-synthesis. We use binding affinity measurements, nanoparticle synthesis, characterization and stabilization assays, and molecular modeling, to investigate a set of sequences based on two known peptides with strong affinity for Au. This set of biomolecules is designed to probe specific sequence and context effects using both point mutations and global reorganization of the peptides. Our data confirm, for a broader range of sequences, that Au nanoparticle/peptide binding affinity alone is not predictive of peptide-mediated colloidal stability. By comparing nanoparticle stabilization assay outcomes with molecular simulations, we establish a correlation between the colloidal stability of the Au nanoparticles and the degree of conformational diversity in the surface-adsorbed peptides. Our findings suggest future routes to engineer peptide sequences for bio-based growth and dispersion of functional nanoparticles in aqueous media. / Air Office of Scientific Research, grant number FA9550-12-1-0226.

Protein-ligand binding and protein allostery play a crucial role in cell signaling, cell regulation, and modern drug discovery. In recent years, experimental studies of protein structures including crystallography, NMR, and Cryo-EM are widely used to investigate the functional and inhibitory properties of a protein. On the one hand, structural classification and feature identification of the structures of protein kinases, HIV proteins, and other extensively studied proteins would have an increasingly important role in depicting the general figures of the conformational landscape of those proteins. On the other hand, free energy calculations which include the conformational and binding free energy calculation, which provides the thermodynamics basis of protein allostery and inhibitor binding, have proven its ability to guide new inhibitor discovery and protein functional studies. In this dissertation, I have used multiple different analysis and free energy methods to understand the significance of the conformational and binding free energy landscapes of protein kinases and other disease-related proteins and developed a novel alchemical-based free energy method, restrain free energy release (R-FEP-R) to overcome the difficulties in choosing appropriate collective variables and pathways in conformational free energy methods like umbrella sampling and metadynamics. / Chemistry