Browsing by Subject "Biophysics"

Electroporation is a biological cell's natural reaction to strong electric fields, where transient pores are created in the cell membrane. While electroporation holds promise of being a safe and effective tool for enhancing molecular delivery in numerous medical applications, it remains largely confined to preclinical research and clinical trials due to an incomplete understanding of the exact mechanisms involved. Muscle fibers are an important delivery target, but traditional theoretical studies of electroporation ignore the individual fiber geometry, making it impossible to study the unique transverse and longitudinal effects from the pulse stimulus. In these long, thin muscle fibers, the total reaction of the fiber to the electric field is due to fundamentally different effects from the constituent longitudinal and transverse components of the electric field generated by the pulse stimulus. While effects from the transverse component have been studied to some degree, the effects from the longitudinal component have not been considered.

This study develops a model of electroporation and delivery of small molecules in muscle tissue that includes effects from both the transverse and longitudinal components of the electric field. First, an asymptotic model of electric potential in an individual muscle fiber is derived that separates the full 3D boundary value problem into transverse and a longitudinal problems. The transverse and longitudinal problems each have their own respective source functions: the new "transverse activating function" and the well known longitudinal activating function (AF). This separation enhances analysis of the different effects from these two AFs and drastically reduces computational intensity. Electroporation is added to the asymptotic fiber model, and simplified two-compartment mass transport equations are derived from the full 3D conservation of mass equations to allow simulation of molecular uptake due to diffusion and the electric field. Special emphasis is placed on choosing model geometry, electrical, and pulsing parameters that are in accordance with experiments that study electroporation-mediated delivery of small molecules in the skeletal muscle of small mammals.

Simulations reveal that for fibers close to the electrodes the transverse AF dominates, but for fibers far from the electrodes the longitudinal AF enhances uptake by as much as 2000%. However, on the macroscopic tissue level, the increase in uptake from the longitudinal AF is no more than 10%, given that fibers far from the electrodes contribute so little to the total uptake in the tissue. The mechanism underlying the smaller effect from the longitudinal AF is found to be unique to the process of electroporation itself. Electroporation occurs on the short time scale of polarization via the transverse AF, drastically increases membrane conductance, and effectively precludes further creation of pores from charging of the membrane via the longitudinal AF. The exact value of enhancement in uptake from the longitudinal AF is shown to depend on pulsing, membrane, and tissue parameters. Finally, simulation results reproduce qualitative, and in some cases quantitative, behavior of uptake observed in experiments.

Overall, percent increase in total tissue uptake from the longitudinal AF is on the order of experimental variability, and this study corroborates previous theoretical models that neglect the effects from the longitudinal AF. However, previous models neglect the longitudinal AF without explanation, while the asymptotic fiber model is able to detail the mechanisms involved. Mechanisms revealed by the model offer insight into interpreting experimental results and increasing efficiency of delivery protocols. The model also rigorously derives a new transverse AF based on individual fiber geometry, which affects the spatial distribution of uptake in tissue differently than predicting uptake based on the magnitude of the electric field, as used in many published models. Results of this study are strictly valid for transport of small molecules through small non-growing pores. For gene therapy applications the model must be extended to transport of large DNA molecules through large pores, which may alter the importance of the longitudinal AF. In broader terms, the asymptotic model also provides a new, computationally efficient tool that may be used in studying the effect of transverse and longitudinal components of the field for other types of membrane dynamics in muscle and nerves.

One of the most significant discoveries from mutational analysis of protein interfaces is that often a large percentage of interface residues negligibly perturb the binding energy upon mutation, while residues in a few critical "hotspots" drastically reduce affinity when mutated. The organization of protein interfaces into hotspots has a number of important implications. For example, small interfaces can have high affinity, and when multiple binding partners are generated to the same protein, they are predisposed to binding the same regions and often have the same hotspots. Even small molecules that bind to interfaces and disrupt protein-protein interactions (PPIs) tend to bind at hotspots. This suggests that some hotspot-forming sites on protein surfaces are intrinsically more apt to form protein interfaces. These observations paint a hotspot-centric picture of PPI energetics, and present a question of fundamental importance which remains largely unanswered: why are hotspots hot?

In order to gain insight into the nature of hotspots I experimentally examined the small, but high-affinity interface between the synthetically evolved ankyrin repeat protein Off7 with E. coli maltose binding protein by characterization of mutant variants and redesigned interfaces. In order to characterize many mutants, I developed two high-throughput assays to measure protein-protein binding that integrate with existing technology for the high-throughput fabrication of genes. The first is an ELISA-based method using in vitro expressed protein for semi-quantitative analysis of affinity. Starting from DNA encoding protein partners, binding data is obtained in just a few hours; no exogenous purification is required. For the second assay, I develop data fitting methods and thermodynamic framework for determination of binding free energies from binding-induced shifts in protein thermal stability monitored with Sypro Orange.

Analysis of Off7/MBP variants using these methods reveals that conservative mutagenesis or local computational repacking is tolerated for many residues in the interface without drastic loss of affinity, except for a single essential hotspot. This hotspot contains a Tyr-His-Asp hydrogen bonding network reminiscent of a common catalytic motif. Substitution of the tyrosine with phenylalanine shows that a single hydrogen bond across the interface is critical for binding. Analysis of the protein database by structural bioinformatics shows that, although rare, this motif is present in other naturally evolved interfaces. Such a triad was found in the homodimeric interface of PH0642 from Pyrococcus horikoshii, and is conserved between many homologues in the nitrilase superfamily, meeting one of the key criteria by which potential hotspots can be identified. This analysis supports a number of analogies between hotspot residues and catalytic residues in enzyme active sites, and raises the intriguing possibility that hotspots may be associated with other structural motifs that could be used for identification or design of PPIs.

Melanin is a biological pigment that is ubiquitous in nature and generally produced within melanosomes, specialized organelles. Typically, melanin is categorized into two distinct classes, based on color and molecular precursor: eumelanin (brown-black) and pheomelanin (yellow-red). Whereas much is known regarding the molecular precursors to the two pigments, an understanding of their resulting molecular structure remains elusive. Despite this lack of knowledge, several functions are attributed to the pigments, including photoprotection and photosensitization. Epidemiological data for skin and ocular cancers have observed an increased incidence for increased relative concentrations of pheomelanin. Furthermore, eumelanin is generally identified as photoprotective and antioxidant, whereas pheomelanin is generally identified as photoreactive and pro-oxidant. This thesis describes the photophysical properties of the naturally-occuring melanin pigments and presents new insights into their roles within the context of skin and ocular cancers.

Photoemission electron microscopy provides a unique opportunity to probe the complex photoproperties of melanins contained within intact melanosomes isolated from tissues of bovine and human eyes. Photoionization threshold potentials characteristic of eumelanin and pheomelanin have been determined and are used to investigate the molecular architecture of the pigments within the melanosome. Furthermore, a novel approach to photoemission electron microscopy is used to obtain the first direct measurements of the absorption coefficients from intact melanosomes.

Human iridal stroma melanosomes are comprised of both eumelanin and pheomelanin in various ratios according to iris color; dark brown and blue-green iris melanosomes are characterized by a eumelanin:pheomelanin ratio of 14.8 and 1.3, respectively. Despite the significant difference in the overall pigment composition, a common eumelanin surface photoionization threshold is obtained for both melanosomes. This data indicates that within the melanosome, the phototoxic pheomelanin pigment is encased by eumelanin. This structure mitigates the adverse photochemical properties of pheomelanin. However, damage to the eumelanic exterior and or significant reduction in the amount of eumelanin present could compromise the protective ability of eumelanin, providing mechanisms for exposure of pheomelanin and consequently contributing to oxidative stress.

The absorption spectra of intact melanosomes of varying melanin compositions were determined over the spectral range from 244 to 310 nm. The absorption spectra of eumelanic melanosomes are similar regardless of monomer composition or embryonic origin. Furthermore, the absorption spectra of melanosomes containing a mixture of pigments were similar to those containing pure eumelanin, arguing that the absorption properties of the melanosome are maintained regardless of increased pheomelanin composition. Therefore, the correlation between epidemiological data and the eumelanin:pheomelanin ratio is not predicted to be a reflection of the melanosome's decreased ability to attenuate biologically relevant wavelengths, but instead is predicted to be a reflection of the different photoreactivities of the melanin pigments contained within.

Molecular recognition is arguably the most elementary physical process essential for life that arises at the molecular scale. Molecular recognition drives events across virtually all length scales, from the folding of proteins and binding of ligands, to the organization of membranes and the function of muscles. Understanding such events at the molecular level is massively complicated by the unique medium in which life occurs: water. In contrast to recognition in non-aqueous solvents, which are driven largely by attractive interactions between binding partners, binding reactions in water are driven in large measure by the properties of the medium itself. Aqueous binding involves the loss of solute-solvent interactions (desolvation) and the concomitant formation of solute-solute interactions. Despite decades of research, aqueous binding remains poorly understood, a deficit that profoundly limits our ability to design effective pharmaceuticals and new enzymes. Particularly problematic is understanding the energetic consequences of aqueous desolvation, an area the Toone and Beratan groups have considered for many years.

In this dissertation, we embark on a quest to shed new light on aqueous desolvation from two perspectives. In one component of this research, we improve current computational tools to study aqueous desolvation, employing quantum mechanics (QM), molecular dynamics (MD) and Monte Carlo (MC) simulations to better understand the behavior of water near molecular surfaces. In the other, we use a synthetic host, cucurbit[7]uril (CB[7]), in conjunction with a de novo series of ligands to study the structure and thermodynamics of aqueous desolvation in the context of ligand binding with atomic precision, a feat hitherto impossible. A simple and rigid macrocycle, CB[7] alleviates the drawbacks of protein systems for the study of aqueous ligand binding, that arise from conformational heterogeneity and prohibitive computational costs to model.

We first constructed a novel host-guest system that facilitates internalization of the trimethylammonium (methonium) group from bulk water to the hydrophobic cavity of CB[7] with precise (atomic-scale) control over the position of the ligand with respect to the cavity. The process of internalization was investigated energetically using isothermal titration microcalorimetry and structurally by nuclear magnetic resonance (NMR) spectroscopy. We show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by an unfavorable desolvation enthalpy of just 0.49±0.27 kcal*mol-1, a value significantly less endothermic than those values suggested from previous gas-phase model studies. Our results offer a rationale for the wide distribution of methonium in biology and demonstrate important limitations to computational estimates of binding affinities based on simple solvent-accessible surface area approaches.

To better understand our experimental results, we developed a two-dimensional lattice model of water based on random cluster structures that successfully reproduces the temperature-density anomaly of water with minimum computational cost. Using reported well-characterized ligands of CB[7], we probed water structure within the CB[7] cavity and identified an energetically perturbed cluster of water. We offer both experimental and computational evidence that this unstable water cluster provides a significant portion of the driving force for encapsulation of hydrophobic guests.

The studies reported herein shed important light on the thermodynamic and structural nature of aqueous desolvation, and bring our previous understanding of the hydrophobic effect based on ordered water and buried surface area into question. Our approach provides new tools to quantify the thermodynamics of functional group desolvation in the context of ligand binding, which will be of tremendous value for future research on ligand/drug design.

Many vital biological tasks, such as protein degradation, DNA strand separation, and viral DNA packaging are performed by ring NTPase assemblies. These assemblies harvest energy from NTP binding and hydrolysis in order to translocate their biopolymer substrate through their central pores. Single-molecule characterization demonstrated that these assemblies are highly coordinated and produce forces an order of magnitude larger than most molecular motors. Recently, many structures of these assemblies have been experimentally solved and resulting globular translocation models have been proposed. While these static structures have provided great insights into how molecular motors assemble, the specific molecular mechanisms that promote, regulate, and coordinate the dynamic translocation processes remain poorly understood. In this dissertation, I use computational tools to model ring ATPase molecular motors in order to elucidate such mechanisms. Initially, I focus on viral packaging ATPases and then generalize my findings to a broader class of motors by studying FtsK-like and AAA+ motors. For all systems, atomistic molecular dynamics simulations were used to calculate free-energy landscapes that predict conformational changes, predict mutual-information-based signaling pathways that couple enzymatic and mechanical activities, predict principal components of motion that describe the enzyme’s native function, and predict the effects of mutagenesis in silico. For viral packaging ATPases, I first predicted that a strictly conserved Walker A arginine residue functions analogously to a sensor II motif arginine found in AAA+ systems, and that it is used to couple ATP binding to lid subdomain rotation. Second, I predicted how mutations in the Walker A and Walker B motifs could abrogate enzyme function. All these predictions were corroborated by collaborators’ extensive experimental characterization. Third, I helped build the first structure of an actively packaging viral ATPase motor into the cryo-EM reconstruction and led the biological interpretation of the resulting structure. Fourth, I used molecular dynamics simulations of pentameric ATPase assemblies to predict how the assemblies respond to nucleotide-occupancy and presence of double-stranded DNA substrate. Based on the structure and simulations, I proposed the helical-to-planar model of viral DNA packaging, which is the first atomistic model that can predict the salient features of viral DNA packaging. Further, this model lays the groundwork of future work by predicting specific conformational changes and interactions that were otherwise obscure from experimental studies. Fifth, I tested a key proposal in my helical-to-planar model by using molecular dynamics simulations to investigate how nucleotide binding is coupled to substrate gripping. The resulting glutamate switch signaling pathway was corroborated by structural data and functional mutagenesis assays. Lastly, I investigated FtsK-like and AAA+ enzymes to probe for molecular mechanisms common to a broad class of translocating ring ATPases. From these studies, I identified a core set of principles that can be modularly added together to describe a number of different translocation models. In summary, the results presented in this dissertation describe fundamental mechanisms of translocating ring ATPase motors. When possible, my computational predictions were corroborated by experimental characterization. When experimental characterization was not yet possible, my predictions and derived models serve as a guide for future studies. The models I derived provide the first comprehensive description of the coordinated conformational changes that drive viral DNA packaging. Further, they have the potential to inform rational design of synthetic molecular motors and anti-viral therapeutics that target the genome packaging step.

Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that in the budding yeast, Saccharomyces cerevisiae, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). I have determined, however, that the Kip1 and Cin8 proteins are actually present at wild-type levels in yeast in the absence of Clb/Cdc28 kinase activity. Here, I show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28, and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, I have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, I propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.

In addition to the positive regulation of kinesin-5 function in spindle assembly, I have also found evidence that suggests CDK phosphorylation of kinesin-5 motors at different sites negatively regulates kinesin-5 activity to prevent premature spindle pole separation. I have also begun to characterize a novel putative role for the kinesins-5 in mitochondrial genome inheritance in S. cerevisiae that may also be regulated by CDK phosphorylation.

In the course of my dissertation research, I encountered problems with several established molecular biology tools used by yeast researchers that I have tried to address. I have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in S. cerevisiae that can be propagated in the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. My new pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed in order to increase the availability of unique restriction sites within their polylinker regions. Hence, my pRSII plasmids are a complete set of integrating, centromere and 2 episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2 and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, I have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin and bialaphos. Finally, in the process of generating the new plasmids, I have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using updated plasmid sequences, I constructed pRS plasmid backbones with a unique restriction site for inserting new markers in order to facilitate future expansion of the pRS/pRSII series.

Many animals can sense the earth’s magnetic field and use it to perform incredible feats of navigation; however, studying this phenomenon in the lab is difficult because behavioral responses to magnetic cues can be highly variable. My Ph.D. research attempts to fill this knowledge gap in the following ways: we first explore potential sources for this variability, including both natural and artificial sources of noise. We then examine the ways in which these natural sources of noise could be used to study magnetoreception in animals that are not feasible to study in the laboratory. Finally, we propose a possible solution for how navigating animals may overcome noise to still accomplish highly accurate migrations. Chapter 1 contains the relevant background and introduction. In Chapter 2, we conduct a synthetic review of natural and anthropogenic sources of radio frequency electromagnetic noise (RF) and its effects on magnetoreception. Anthropogenic RF has been shown to disrupt magnetic orientation behavior in some animals. Two sources of natural RF might also have the potential to disturb magnetic orientation behavior under some conditions: solar RF and atmospheric RF. In this review, we outline the frequency ranges and electric/magnetic field magnitudes of RF that have been shown to disturb magnetoreceptive behavior in laboratory studies and compare these to the ranges of solar and atmospheric RF. Frequencies shown to be disruptive in laboratory studies range from 0.1 to 10 MHz, with magnetic magnitudes as low as 1 nT reported to have effects. Based on these values, it appears unlikely that solar RF alone routinely disrupts magnetic orientation. In contrast, atmospheric RF does sometimes exceed the levels known to disrupt magnetic orientation in laboratory studies. We provide a reference for when and where atmospheric RF can be expected to reach these levels, as well as a guide for quantifying RF measurements.

In Chapter 3, we explore how these natural sources of noise may allow us to study magnetoreception in animals that are not feasible to study in the laboratory. Although it is difficult to perform behavioral experiments on baleen whales, it may be possible to use live stranding data (strandings that indicate the whale may have made a navigational error, rather than those having died at sea and washed ashore) as a tool to investigate the cues they use while navigating. Here we show that there is a 2.1-fold increase in the likelihood of a live gray whale (Eschrichtius robustus) stranding (n=186) on days with a high sunspot count than on low sunspot days (p<0.0001). Increased sunspot count is strongly correlated with solar storms – sudden releases of high-energy particles from the sun which have the potential to disrupt magnetic orientation behavior when they interact with earth’s magnetosphere. We further explore this relationship by examining portions of earth’s electromagnetic spectrum that are affected by solar storms and found a 3.7-fold increase in the likelihood of a live stranding on days with high solar radio flux (RF) as measured from earth (p<0.0001). One hypothesized mechanism for magnetoreception, the radical-pair theory, predicts that magnetoreception can be disrupted by RF radiation, and RF noise has been shown to disrupt magnetic orientation in certain species. To our knowledge, this is the first evidence that provides support for a specific magnetoreception mechanism in whales.

Finally, in Chapter 4, we propose a mechanism for how magnetoreceptive animals may overcome noise to perform incredibly accurate migrations. Many animals use the geomagnetic field to migrate long distances with high accuracy; however, research has shown that individual responses to magnetic cues in the laboratory can be highly variable. Thus, it has been hypothesized that magnetoreception alone is insufficient for accurate migrations and animals must either switch to a more accurate sensory cue or integrate their magnetic sense over time. Here we suggest that magnetoreceptive migrators could also use collective navigation strategies. Using agent-based models, we compare agents utilizing collective navigation to both the use of a secondary sensory system and time-integration. In our models, by using collective navigation alone, over 70% of the group is still able to successfully reach their goal even as their ability to navigate becomes extremely noisy. To reach the same success rates, in our models, a secondary sensory system must provide perfect navigation for over 73% of the migratory route, and time integration must integrate over 50 time-steps, indicating that magnetoreceptive animals could benefit from using collective navigation. Finally, we explore the impact of population loss on animals relying on collective navigation. We show that as population density decreases, a greater proportion of individuals fail to reach their destination and, in our models, a 50% population reduction resulted in up to a 37% decrease in the proportion of individuals completing their migration. We additionally show that this process is compounding, eventually resulting in complete population collapse.

Biological signaling via DNA-mediated charge transfer between high-potential [4Fe4S]2+/3+ clusters is widely discussed in the literature. Recently, it was proposed that for DNA replication on the lagging strand, primer handover from primase to polymerase α is facilitated by DNA-mediated charge transfer between the [4Fe4S] clusters housed in the respective C-terminal domains of the proteins. Using a theoretical-computational approach, I established that redox signaling between the clusters in primase and polymerase α cannot be accomplished solely by DNA-mediated charge transport, due to the unidirectionality of charge transfer between the [4Fe4S] cluster and the nucleic acid. I extended the study by developing an open-source electron hopping pathway search code to characterize hole hopping pathways in proteins and nucleic acids. I used this module to analyze protective hole escape routes in cytochrome p450, cytochrome c oxidase, and benzylsuccinate synthase. Next, I used the module to analyze molecular dynamics snapshots of a mutant primase, where the Y345C mutation (found in gastric tumors) attenuates charge transfer between the [4Fe4S] cluster and nucleic acid, which in turn, could disrupt the signaling process between primase and polymerase α. In another protein-nucleic acid system, I found that charge transfer in the p53-DNA complex plays an important role for p53 to differentiate Gadd45 DNA and p21 DNA in metabolic pathway regulation. Using density functional theory calculations on molecular dynamics snapshots, I found that hole transfer (HT) from Gadd45 DNA to the proximal cysteine residue in the DNA-binding domain of p53 is preferred over HT from p21 DNA to cysteine. This preference ensures that the p21 DNA remains bound to the transcription factor p53 which induces the transcription of the gene under cellular oxidative stress. This dissertation concludes with a study that demonstrates similar electron conductivities between an artificial nucleic acid, 2'-deoxy-2'-fluoro-arabinonucleic acid (2’F-ANA), and DNA. Compared to DNA, 2’F-ANA offers the additional benefit of chemical stability with respect to hydrolysis and nuclease degradation, thereby promoting its use as a sensor in biological systems and cellular environments.

Embryonic development involves a precisely orchestrated interplay between gene expression, tissue movement and cell-shape changes. Using time-lapsed in vivo confocal microscopy we investigate the time-dependence of cell-shape changes for essentially all of the cells in the amnioserosa tissue during the early-to-late stages of dorsal closure in Drosophila melanogaster. Dorsal closure is a critical stage during embryogenesis, where two epidermal tissues are brought together by the force producing machinery in the surrounding tissues, including the amnioserosa tissue. The environment these cells exist in is dominated by viscous forces, making the observable kinematics the result of active contractile force imbalances along the cell peripheries. Our image contrast is due to GFP-DE-cadherin, a relatively bright fluorescent construct that localizes at cell-cell junctions. Using custom written segmentation software we quantify cell apical areas from confocal images. By considering the kinematics of individual cells we investigate the forces produced by the amnioserosa tissue. We confirm previous observations of area pulsations or oscillations and that, within the dorsal opening, areas of peripheral amnioserosa cells are smaller than the areas of interior cells [Fernandez et al., 2007, Gorfinkiel et al., 2009, Solon et al., 2009, Blanchard et al., 2010]. In addition to oscillations, we find that cells in the low-Reynolds environment of the amnioserosa tissue exhibit ingression processes, a persistent loss of apical area resulting in the internalization of the cell. We develop an empirical model that quantifies the kinematics of the ingression processes of a substantial fraction of the amnioserosa cells. We also account for these observations with a biophysical model that quantifies the (spatially averaged) net force from experimental data and explicitly treats the dynamics of oscillations and ingression. Utilizing both models, we find that approximately half of the amnioserosa cells exhibit a loss of apical cross-sectional area dominated by an irreversible ingression process. For these cells, a transition is resolved from largely reversible oscillations to the onset of an ingression process. We also investigate variability in cell kinematics according to location within the dorsal opening and we find that cells ingress along each leading edge, in addition to previously observed ingression associated with the zipping process or associated with apoptosis. We attribute cell-to-cell variability in the maximum rate of constriction during the ingression processes to be a consequence of variability in the magnitude of force produced by the cytoskeleton. Finally, we investigate invariant properties, i.e., time-independent, global properties of dorsal closure and find nearly constant rates of completion of ingression processes as well as a constant of proportionality that relates the area of the dorsal opening to its two principle axes.

For over 200 years, cell function and behavior has been the subject of significant interest. Although microscopic, mammalian cells are fantastically complicated and need to overcome tremendous environmental inertia to maintain homeostasis, such as facilitating ion gradients and intracellular transport, the movement of cargo through a viscous, crowded cytosol. This latter point is especially important because many diseases, including Alzheimer’s disease, are associated with aberrant transport. Thus, determining how a cell responds to environmental stimuli is critical to building an understanding of fundamental biophysics and working toward future curative measures for transport-related disease.This dissertation begins with a high-level, epidemiological perspective on the co-occurrence of pulmonary diseases in the United States with pneumoconiosis to contextualize microscopic cell responses to damage with macroscopic outcomes. Pneumoconiosis is caused by inhaled dusts and nanomaterials, which can become embedded in and inflame lung tissues. We found that pneumoconiosis is associated with increased rates of chronic obstructive pulmonary disease (COPD), lung cancer, and pneumonia at time of death. When we combined pneumoconiosis diagnoses with the known covariate of smoking history, smokers with pneumoconiosis had the highest rate of COPD in our data, indicating a potential synergistic effect of lung damage. Interestingly, we found that smoking history and pneumoconiosis were more associated with lung cancer and pneumonia, respectively. Through presented case studies in non-mining/construction occupations, we note that specific pneumoconioses can occur on local scales, demonstrating that even if nanomaterials are too varied to appear in an aggregate population, the risks of increased disease rate as a result of microscopic lung injury are still present. This is essential for future regulation and policy decisions as nanomaterial production continually increases. To further explore microscopic cell responses to controlled stimuli, we used particle tracking microscopy to follow trafficked organelles and evaluate how the cell uses intracellular transport and reacts to disruptions. The classic approach to analyzing particle tracking data is the mean squared displacement (MSD). Despite its ubiquity, recent work has shown that the MSD is a flawed method because it is unstable with respect to noise, curve fitting choices, and observation window. Here, we present a novel tracking framework that uses a Bayesian changepoint segmentation strategy and then infers population motility from segment velocities. This method avoids the use of MSDs and is efficient and stable in response to trajectories of different quality and length. We demonstrate this software on tracked lysosomes in epithelial cells. We found that there is a clear difference in the frequency of motion for lysosomes depending on where in the cell they were located, with lysosomes in the perinuclear region moving less often than those in the periphery. This is an extremely important finding because it robustly distinguishes these two regions over thousands of lysosome observations and much of the current particle tracking literature ignores region as a factor, potentially exposing any results to selection bias. Separately, we found that the size of lysosomes, which was controlled with sucrose-induced osmotic swelling, had no effect on transport frequency; however, the speed of large lysosomes was slower than with small lysosomes. Next, we generalize our tracking system to include all vesicles, rather than only lysosomes. With these conditions, we present an exciting new result: disruption of the endoplasmic reticulum with palmitate, a fatty acid found at elevated levels in patients with diabetes and obesity, significantly decreases vesicle motility. This effect was independent of any reduction in ATP levels or cell viability and appears to be associated with the distortion of the ER we observed under these conditions. This result points to areas of future research in the biophysical complications associated with these diseases and further underscores recent work detailing the extensive interactions between the ER and endocytic vesicles. Paired with this analysis, we also observed that macromolecular crowding has no effect on directed transport through the reduction of ribosome concentration, indicating that directed intracellular transport is quite efficient despite significant obstacles in the cytosol. Looking further at the cytoskeleton, we show that disruption of actin filaments and microtubules both decrease vesicle motility as expected. However, we found that disruption of intermediate filament organization with withaferin A significantly decreases vesicle motility in a dose-dependent fashion. Unlike microtubules and actin filaments, there are no molecular motors associated with intermediate filaments, so this result may be tied to cytoskeletal interactions and merits further exploration. In summary, this dissertation details analyses that explore and characterize disruptions to cellular homeostasis. We first provide an updated perspective of dust inhalation diseases in the United States to provide context for cell damage on a macroscopic level and advocate for intentional regulation of nanomaterials as production and exposure risk increase. We also demonstrate an effective Bayesian particle tracking analysis alternative to the mean squared displacement, which overcomes the latter’s limitations. We then use this new tool to learn significant new information about intracellular transport, particularly that there are regional distinctions in vesicle behavior and that ER disruption with palmitate causes a dramatic decrease in transport without affecting viability. Overall, we are most excited for the potential for this analysis to be used across a variety of problems and disciplines and look forward to its implementation.

This dissertation research focused on the ways in which clear crustaceans with complex bodies (i.e. with hard cuticles, thick muscles, and other internal organs) maintain transparency across their entire body volume. I used transparent crustacean species that had relatively large (> 25 mm long and > 2 mm thick) bodies and that occupied physically different (pelagic vs. benthic reef) habitats. Studying these transparent crustaceans and making comparisons with closely related opaque crustaceans provided some of the first insights into the puzzling problem of the physical basis of transparency in whole organisms.

First, I examined the ultrastructure of the cuticle of hyperiid amphipods, the first surface to interact with light, to understand what features may minimize reflectance. I investigated the cuticle surfaces of seven species of mostly transparent hyperiids using scanning electron microscopy and found two previously undocumented features that reduced reflectance. I found that the legs of Cystisoma spp. were covered with an ordered array of nanoprotuberances that functioned optically as a gradient refractive index material to reduce reflections. Additionally, I found that Cystisoma and six other species of hyperiids were covered with a monolayer of homogenous nanospheres (approximately 50 nm to 350 nm in diameter) that were most likely bacteria. Optical modeling demonstrated that both the nanoprotuberances and the monolayers reduced reflectance by as much as 250-fold. Even though the models only considered surface reflectance and not internal light scattering, these models showed that the nanoprotuberances and spheres could improve crypsis in a featureless habitat where the smallest reflection could render an animal vulnerable to visual predation.

Second, I took a morphological approach to investigate how light scattering may be minimized internally. Using bright field microscopy, I explored whether there were any gross anatomical differences in the abdominal muscles between a transparent species of shrimp, Ancylomenes pedersoni, and a similarly sized opaque shrimp species, Lysmata wurdemanni. I found no differences in muscle fiber size or any other features. Using transmission electron microscopy (TEM) to visualize muscle ultrastructure, I found that the myofibrils of the transparent species were twice the diameter of the opaque species (mean values of 2.2 μm compared to 1.0 μm). Over a given distance of muscle, light passes through fewer myofibrils due to their larger diameter, with fewer opportunities for light to be scattered at the interfaces between the high-index myofibrillar lattice and the surrounding lower-index fluid-filled sarcoplasmic reticulum (SR). Additionally, because transparency is not always a static trait and can sometimes be disrupted after exercise or physiological stress, I compared the ultrastructure of muscle in transparent A. pedersoni shrimp with the ultrastructure of muscle in A. pedersoni that had temporarily turned opaque after exercise. I found that in this opacified tissue, the fluid-filled space around myofibrils had an increased thickness of 360 nm as compared to a normal thickness of 20 nm. While this could have been a fixation artifact, this result still suggests that opacified tissue had some change in osmolarity or increase in fluid. Models of light scattering across a range of thicknesses and possible refractive indices showed that this observed increase in fluid-filled space dramatically reduced transparency.

Third, I further investigated how exertion or physiological stress may disrupt transparency, what may occur in the tissues to cause this disruption, and what may explain the increased fluid-filled SR interface. I hypothesized that increased perfusion, or an increase in blood volume between muscle fibers, can disrupt the normal organization of tissue, resulting in increased light scattering. I measured pre- and post-exercise perfusion via the injection of a specific fluorescent stain (Alexa Fluor 594-labeled wheat germ agglutinin) that labeled the sarcolemmal areas in contact with hemolymph and the endothelial cells of the blood vessels, and found more open vessels and greater hemolymph perfusion around fibers post-exercise. Changing salinity in the shrimps’ tanks, wounding the shrimp, and injecting proctolin (a vasodilator) were also associated with increased opacity and perfusion. To visualize the shrimps’ overall muscle morphology, I used Diffusible Iodine-based Contrast-Enhanced Computed Tomography (DICECT) to scan one control (transparent) and one experimental (opaque) A. pedersoni. The resulting images added further support to my hypothesis that hemolymph volume in the muscle increases in post-exercise opacified A. pedersoni.

Despite the best efforts of both academia and the pharma industry, most non-resectable cancers remain uncurable and lethal. The world health organization (WHO) believes cancer to be the second leading cause of death worldwide, with roughly 9.6 million deaths in 2018. Meanwhile, the emergence of antimicrobial resistance (AMR), or superbugs, is an increasingly large medical crisis, with estimates as high as 700,000 deaths for 2018 worldwide. This number is increasing rapidly. These unmet medical needs, although distinct, are intimately related by the need for better chemistry and intelligent drug design.

Both AMR and cancer could benefit from the expansion of the druggable proteome through the inhibition of protein-protein interactions (PPIs). PPIs drive both intra- and inter-cellular communication, and therefore their inhibition is vital for disease modulation. Moreover, both AMR and cancer therapeutics suffer from the rapid emergence of drug resistance. Even great drugs that function perfectly at first frequently lose effectiveness a few months later, due to the rapid emergence of drug resistance.

Here, I discuss my contributions towards developing a PPI inhibitor to KRas, the most commonly activated oncogene in cancer. Through the use of OSPREY, a state-of-the-art computational protein and drug design (CPDD) software, and using KRas’ native ligand Raf-1 RBD as a starting point, we developed a super-binder with single-digit nanomolar affinity for KRas. The development and validation of this biologic inhibitor required the development of four novel biochemical assays to study binding to KRas and the inhibition of the KRas:Raf interaction.

I also discuss my contributions towards enhancing our ability to predict resistance mutations through the use of OSPREY. This work focused on novel mechanisms of resistance in the dihydrofolate reductase of Staphylococcus aureus (SaDHFR). Specifically, we investigated the role of plasmid-borne resistance genes in Staph, as well as the mechanism of resistance due to the emergence of the F98Y and V31L resistance mutations. We discovered a potential new mechanism of resistance based on the formation of a tricyclic NADPH configuration, which we have named chiral evasion.

Finally, I discuss lessons learned from benchmarking OSPREY and share observations that can be used by drug designers using CPDD tools to enhance the accuracy and predictive potential of their results.

In conclusion, a combination of OSPREY and biochemical assays was used towards overcoming two of the largest limitations in drug development that directly affect global human health: the development of PPI inhibitors and overcoming drug resistance. We identified a novel hot-spot in the KRas:Raf interface that can successfully be used to optimize the PPI and develop a biologic inhibitor to KRas. We generated models that explain the mechanism of inhibition of both V31L and F98Y in the context of chiral evasion through a tricyclic NADPH configuration, and we benchmarked OSPREY and observed features that can contribute towards the predictive accuracy of CPDD tools.

Proteins are involved in all of life's processes and are also responsible for many diseases. Thus, engineering proteins to perform new tasks could revolutionize many areas of biomedical research. One promising technique for protein engineering is computational structure-based protein design (CSPD). CSPD algorithms search large protein conformational spaces to approximate biophysical quantities. In this dissertation we present new algorithms to realistically and accurately model how amino acid mutations change protein structure. These algorithms model continuous flexibility, protein ensembles and positive/negative design, while providing guarantees on the output. Using these algorithms and the OSPREY protein design program we design and apply protocols for three biomedically-relevant problems: (i) prediction of new drug resistance mutations in bacteria to a new preclinical antibiotic, (ii) the redesign of llama antibodies to potentially reduce their immunogenicity for use in preclinical monkey studies, and (iii) scaffold-based anti-HIV antibody design. Experimental validation performed by our collaborators confirmed the importance of the algorithms and protocols.

Small-angle X-ray scattering (SAXS) is a biophysical technique that allows one to study the statistical conformation of a biopolymer in solution. The two-dimensional data obtained from SAXS is a low-resolution probe of the statistical conformation- it is a population weighted orientational average of all conformers within a conformational ensemble. Traditional biological SAXS experiments seek to describe an "average" structure of a protein, or enumerate a "minimal ensemble" of a protein at the atomic resolution scale. However, for highly flexible proteins, an average structure or minimal ensemble may be insufficient for enumeration of conformational space, and may be an over-parameterized model of the statistical conformation. This work describes a SAXS analysis of highly flexible proteins and presents a protocol for describing the statistical conformation based on minimally parameterized polymer physics models and judicious use of ensemble modeling. This protocol is applied to the structural characterization of S. aureus protein A - a crucial virulence factor - and Fibronectin III domains 1-2 - an important structural protein.

This study is focused on designing and characterizing protein-based building blocks in order to construct self-assembled nano-structured biomaterials. In detail, this research aims to: (1) investigate a new class of proteins that possess nanospring behaviors at a single-molecule level, and utilize these proteins along with currently characterized elastomeric proteins as building blocks for nano-structured biomaterials; (2) develop a new method to accurately measure intermolecular interactions of self-assembling two or more arbitrary (poly)peptides, and select some of them which have appropriate tensile strength for crosslinking the proteins to construct elastomeric biomaterials; (3) construct well-defined protein building blocks which are composed of elastomeric proteins terminated with self-oligomerizing crosslinkers, and characterize self-assembled structures created by the building blocks to determine whether the elasticity of proteins at single-molecule level can be maintained.

Primary experimental methods of this research are (1) atomic force microscope (AFM) based single-molecule force spectroscopy (SMFS) that allows us to manipulate single molecules and to obtain their mechanical properties such as elasticity, unfolding and refolding properties, and force-induced conformational changes, (2) AFM imaging that permits us to identify topology of single molecules and supramolecular structures, and (3) protein engineering that allows us to genetically connect elastomeric proteins and self-assembling linkers together to construct well-defined protein building blocks.

Nanospring behavior of á-helical repeat proteins: We revealed that á-helical repeat proteins, composed of tightly packed á-helical repeats that form spiral-shaped protein structures, unfold and refold in near equilibrium, while they are stretched and relaxed during AFM based SMFS measurements. In addition to minimal energy dissipation by the equilibrium process, we also found that these proteins can yield high stretch ratios (>10 times) due to their packed initial forms. Therefore, we, for the first time, recognized a new class of polypeptides with nanospring behaviors.

Protein-based force probes for gauging molecular interactions: We developed protein-based force probes for simple, robust and general AFM assays to accurately measure intermolecular forces between self-oligomerization of two or more arbitrary polypeptides that potentially can serve as molecular crosslinkers. For demonstration, we genetically connected the force probe to the Strep-tag II and mixed it with its molecular self-assembling partner, the Strep-Tactin. Clearly characterized force fingerprints by the force probe allowed identification of molecular interactions of the single Strep-tag II and Strep-Tactin complex when the complex is stretched by AFM. We found a single energy barrier exists between Strep-tag II and Strep-Tactin in our given loading rates. Based upon our demonstration, the use of the force probe can be expanded to investigate the strength of interactions within many protein complexes composed of homo- and hetero-dimers, and even higher oligomeric forms. Obtained information can be used to choose potential self-assembling crosslinkers which can connect elastomeric proteins with appropriate strength in higher-order structures.

Self-assembled nano-structured biomaterials with well-defined protein-based building blocks: We constructed well-defined protein building blocks with tailored mechanical properties for self-assembled nano-structured materials. We engineered protein constructs composed of tandem repeats of either a I27-SNase dimer or a I27 domain alone and terminated them with a monomeric streptavidin which is known to form extremely stable tetramers naturally. By using molecular biology and AFM imaging techniques, we found that these protein building blocks transformed into stable tetrameric complexes. By using AFM based SMFS, we measured, to our knowledge for the first time, the mechanical strength of the streptavidin tetramer at a single-molecule level and captured its mechanical anisotropy. Using streptavidin tetramers as crosslinkers offers a unique opportunity to create well-defined protein based self-assembled materials that preserve the molecular properties of their building blocks.

One of the main challenges that biological oscillators face at the cellular level is maintaining coherence in the presence of molecular noise. Mechanisms of noise resistance have been proposed, however the findings are sometimes contradictory and not universal. Another challenge faced by biological oscillators is the proper timing of cellular events and effective distribution of cellular resources when there is more than one oscillator in the same cell. Biological oscillators are often coupled, however, the mechanisms and extent of these couplings are poorly understood. In this thesis, I describe three separate yet interconnected projects in an attempt to understand these biophysical phenomena.

I show that slow DNA unbinding rates are important in titration-based oscillators and can mitigate molecular noise. Multiple DNA binding sites can also increase the coherence of the oscillations through protected states, where the DNA binding/unbinding between these states has little effect on gene expression. I then show that experimental titration-based oscillator in budding yeast is innately coupled to the cell cycle. The oscillator and the cell cycle show 1:1 and 2:1 phase locking similar to what has been observed in natural systems. Finally, by studying the relationship between the circadian redox rhythm and genetic circadian clock in plants I show how perturbation of one of the coupled oscillators can be transformed into a reinforcement signal for the other one via a balanced network architecture.

Biological macromolecules can rearrange interdomain orientations when binding to various partners. Interdomain dynamics serve as a molecular mechanism to guide the transitions between orientations. However, our understanding of interdomain dynamics is limited because a useful description of interdomain motions requires an estimate of the probabilities of interdomain conformations, increasing complexity of the problem.

Staphylococcal protein A (SpA) has five tandem protein-binding domains and four interdomain linkers. The domains enable Staphylococcus aureus to evade the host immune system by binding to multiple host proteins including antibodies. Here, I present a study of the interdomain motions of two adjacent domains in SpA. NMR spin relaxation experiments identified a 6-residue flexible interdomain linker and interdomain motions. To quantify the anisotropy of the distribution of interdomain orientations, we measured residual dipolar couplings (RDCs) from the two domains with multiple alignments. The N-terminal domain was directly aligned by a lanthanide ion and not influenced by interdomain motions, so it acted as a reference frame to achieve motional decoupling. We also applied {\it de novo} methods to extract spatial dynamic information from RDCs and represent interdomain motions as a continuous distribution on the 3D rotational space. Significant anisotropy was observed in the distribution, indicating the motion populates some interdomain orientations more than others. Statistical thermodynamic analysis of the observed orientational distribution suggests that it is among the energetically most favorable orientational distributions for binding to antibodies. Thus, the affinity is enhanced by a pre-posed distribution of interdomain orientations while maintaining the flexibility required for function.

The protocol described above can be applied to other biological systems in general. Protein molecule calmodulin and RNA molecule trans-activation response element (TAR) also have intensive interdomain motions with relative small intradomain dynamics. Their interdomain motions were studied using our method based on published RDC data. Our results were consistent with literature results in general. The differences could be due to previous studies' use of physical models, which contain assumptions about potential energy and thus introduced non-experimental information into the interpretations.

Monomolecular hybrid organic-inorganic interfaces provide opportunities for applications in fields ranging from sensors to electronics. In this thesis, we report our efforts towards (1) developing a universal method for the modification and soft-lithographic patterning of inorganic materials with stable and functional organic systems; and (2) apply our surface fabrication techniques to advance our understanding of molecular recognition force microscopy.

We report the development of a novel bi-layered molecular system that, in conjunction with an inkless catalytic microcontact printing technique, can be used to accurately replicate micro- and nano-scale patterns of chemically distinctive reactive functionalities on virtually any surface, including inorganic semiconductors. Catalytic printing alleviates problems associated with ink diffusion and enables high resolution replication of patterns through specific chemical or biochemical reaction between a functional surface and a stamp-immobilized catalyst. The methodology provides precise control over shape and size of pattern features and provides access to chemically discriminated patterns that can be further functionalized with organic and biological molecules. We demonstrate catalytic printing on both oxide-free silicon and germanium, substrates that do not react readily with organic molecules and have not heretofore been patterned through traditional approaches. Our approach we relies on a stable highly ordered bilayered molecular system that both affords complete protection of all surface-exposed inorganic atoms with stable covalent bonds and supports covalent immobilization of a reactive overlayer, yielding stability and functionality to the surface. A catalytic acidic stamp was used to achieve pattern-specific hydrolysis of N-hydroxysuccinimide-activated acids immobilized on Si and Ge. Further modification of the chemically discriminated patterns enables chemoselective anchoring of organic molecules and protein.

We demonstrated the utility of the strategy towards a variety of inorganic oxides, including ITO. Utilizing the functionalized bi-layered system on ITO, a single molecular system in combination with different printing approaches can be used to immobilize multiple organic functionalities with exquisite spatial control. The system was used to investigate structure - function relationships of the ordered and functional molecular system on ITO to vertically and laterally control charge injection in organic light emitting diodes (OLEDs).

Finally, we report fabrication of functional hybrid organic-inorganic interfaces for the study of immobilized binding partners, lactose-g3 and complementary ssDNA, in molecular recognition force microscopy (MRFM). We use our system to evaluate the effect of contact force on specific interactions and the effect of dwell time and tether length on the probability of ligand-receptor binding. The methodologies developed enable a reliable evaluation of thermodynamic parameters using MRFM.

The identification and quantification of protein-protein interactions in large scale is critical to understanding biological processes at a systems level. Current approaches for the analysis of protein -protein interactions are generally not quantitative and largely limited to certain types of interactions such as binary and strong binding interactions. They also have high false-positive and false-negative rates. Described here is the development of and application of mass spectrometry-based proteomics metehods to detect and quantify the strength of protein-protein and protein-ligand interactions in the context of their interaction networks. Characterization of protein-protein and protein-ligand interactions can directly benefit diseased state analyses and drug discovery efforts.

The methodologies and protocols developed and applied in this work are all related to the Stability of Unpurified Proteins from Rates of amide H/D Exchange (SUPREX) and Stability of Protein from Rates of Oxidation (SPROX) techniques, which have been previously established for the thermodynamic analysis of protein folding reactions and protein-ligand binding interactions. The work in this thesis is comprised of four parts. Part I involves the development of a Histidine Slow H/D exchange protocol to facility SURPEX-like measurements on the proteomic scale. The Histidine Slow H/D exchange protocol is developed in the context of selected model protein systems and used to investigate the thermodynamic properties of proteins in a yeast cell lysate.

In Part II an isobaric mass tagging strategy is used in combination with SPROX (i.e., a so-called iTRAQ-SPROX protocol) is used to characterize the altered protein interactions networks associated with lung cancer. This work involved differential thermodynamic analyses on the proteins in two different cell lines, including ADLC-5M2 and ADLC-5M2-C2.

Parts III and IV of this thesis describe the development and application of a SPROX protocol for proteome-wide thermodynamic analyses that involves the use of Stable Isotope Labeling by Amino acid in cell Culture (SILAC) quantitation. A solution-based SILAC-SPROX protocol is described in Part III and a SILAC-SPROX protocol involving the use of cyanogen bromide and a gel-based fractionation step is described in Part IV. The SILAC-SPROX-Cyanogen bromide (SILAC-SPROX-CnBr) protocol is demonstrated to significantly improve the peptide and protein coverage in proteome-wide SPROX experiments. Both the SILAC-SPROX and SILAC-SPROX-CnBr porotocols were used to characterize the ATP binding properties of yeast proteins. Ultimately, the two protocols enabled 526 yeast proteins to be assayed for binding to AMP-PNP, an ATP mimic. A total of 140 proteins, including 37 known ATP-binding proteins, were found to have ATP binding interactions.