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Many processes in physics and biology can be understand through the framework of escape from a metastable state, including (but not limited to) the rates of chemical reactions, the unfolding of proteins, the nucleation of bubbles, and the condensation of gases. To understand the kinetics of these processes, we have to be able to calculate the rate of escape. In this thesis, I solve several of such of escape problems, each addressing a specific physical or biological system. I first show how the forced unfolding of heteropolymers could be a process with non-exponential kinetics, developing ideas about the importance of unfolding pathways in determining kinetics of unfolding. Then, I consider forced unfolding when a molecule is attached to a yielding (viscoelastic) substrate, and a constant force is applied. I show that the rates of unfolding depend on both the elastic and viscous response of the substrate. This problem is related to the biological process of mechanosensing, when the unfolding `sensor' protein exposes catalytic residues and generates a chemical signal to the cell. Related to this is the analysis of population-dynamics study of cells adhesion on substrates, which allows me to extract key characteristics and parameters of the adhesome complex. Then, I apply the ideas of escape from a metastable state to ask about the rates of a ligand at the end of a tethered polymer binding to a surface receptor, using a mean field approach to reduce the problem to one dimension. I show that there is a trade-off between the entropic cost of reaching to a receptor vs the volumetric cost of expanding the tether length. I then show that for a Gaussian chain with multiple ligands along its length, there exists a finite, non-zero optimal number of ligands to minimise the time taken for the end of the chain to bind to the surface. Finally, I consider the problem of microswimmers in an obstacle lattice, calculating their transport properties, and showing how we can use lattices to examine the underlying stochastic dynamics.

Biopolymers, such as proteins and nucleic acids, serve important functions dictated by their structural folding. Information excavated from the intricate structural arrangements of biopolymers have been proved to be vital in diagnosing diseases, unveiling disease mechanisms, and developing new drugs. A continuous effort has been put to improve the characterization techniques of these biomolecules so that more precision can be achieved in terms of detailing their structure-function relationship. Mass spectrometry (MS) is one such technique that has become very relevant for structural biology with the emergence of soft ionization techniques such as electrospray ionization (ESI). A prerequisite to the application of ESI-MS for structural investigation of biopolymers is that the tertiary or quaternary structural components need to be retained in the gas phase of MS. The subdivision of biological mass spectrometry that employs techniques and practices to preserve the native structures of biomolecules in the gas phase is now largely known as ‘native MS’. With the growing complexity of the targeted analytes, native MS promotes the inclusion of techniques that are more capable of deciphering the dynamic structural behavior of biopolymers. For example, proteins that are of intractable (very flexible) nature or that exist in multiple conformations in solution, require an MS-based approach beyond regular ESI to address these challenges. A new spray-based ionization technique namely capillary Vibrating Sharp-edge Spray Ionization (cVSSI) is demonstrated to provide improved field-free ionization capability to promote conformer preservation in the gas phase of MS, especially for the proteins possessing greater conformational flexibility in solution. Two such proteins, leptin and thioredoxin, exhibit bimodal charge state distributions (CSDs) upon the application of voltage; only a monomodal distribution is observed for field-free conditions of cVSSI. In addition, a multidevice cVSSI approach is demonstrated to achieve in-droplet hydrogen/deuterium exchange to directly probe the solution conformer heterogeneity of a well-studied protein, ubiquitin. The presence of co-existing protein solution structures under native and denaturing solution conditions have been distinguished by the isotopic distributions of deuterated ubiquitin ions. The interchange of the relative presence of the conformers within the ensembles corresponds accordingly with the degree of denaturants (methanol) added. Finally, cVSSI is demonstrated as an ionization platform to perform controlled unfolding/refolding of ubiquitin in the droplets by adding voltage sequentially. The mass spectral features are monitored carefully to detail the impacts of voltage related to structural transformations, adducts, and multimers of ubiquitin.

Protein misfolding and self-assembly are important in a variety of processes in functional and dysfunctional biology, as well as in nanotechnology. In all these contexts, it is crucial to understand the molecular mechanisms underlying protein unfolding, aggregation or phase transition. Performing experiments at different temperatures is a conventional, powerful method to extract information about the kinetics and thermodynamics of these processes. However, standard bulk techniques can suffer from limitations related for instance to the amount of sample required, low throughput, and extensive manual handling. Herein, we develop new methods that integrate droplet-based microfluidics with label-free detection techniques and external components that allow an accurate control of temperature on-chip. We show the potential of these devices in increasing our molecular understanding of different processes: i) the kinetics of rapid biomolecular events occurring on a time scale of milliseconds and seconds, including amyloid elongation and enzyme unfolding; ii) thermodynamic and kinetic aspects of phase separation of polymers and proteins. Since phase separation is not an intrinsic property of the molecule but depends also on buffer composition, we propose a continuous-flow device for formulation screening, which automatically displaces and mixes reagents based on valves and elastomeric pumps. We demonstrate the power of the device by analyzing the effect of intrinsic and extrinsic factors on the phase separation of model thermoresponsive polymers, identifying conditions that lead to advanced functions such as a reentrant phase. Finally, to extend the application of these devices to the analysis of aggregation in complex biological mixtures, we select probes recently developed in the context of Alzheimer’s imaging and explore their potential for the specific and sensitive staining of amyloids. Overall, these results show the potential of droplet-based and valve-based microfluidic technology to study polymer and protein self-assembly, thanks to the integration with external components and the label-free readout possibilities that it offers.

Protein quality control is one of the key cellular activities in any living cell, as it provides folding assistance or degradation mechanisms to prevent undesirable off-pathway reactions such as misfolding or aggregates using molecular chaperones. Bacterial Caseinolytic proteases (Clp) promote the degradation pathways by unfolding and translocating tagged substrate proteins (SPs) using powerful ring-shaped AAA+ (ATPases Associated with diverse cellular Activities) motor component with a narrow central pore and later deliver the unfolded polypeptide chain into the peptidase chamber for ultimate destruction into small peptide fragments. ATP fueled conformational transitions of subunits generate a repetitive mechanical unfolding force at the central pore loops and apply this force onto transient residues of SP that reside at the central pore and promote the unfolding and translocation mechanisms. Some aspects of these processes are addressed in experimental and computational studies. Nevertheless, the effects of mechanical anisotropies, non-native contacts, and topological features of SPs on allosteric cycle coupled Clp-mediated unfolding and translocation of SPs into peptidase chamber still remain unclear. To answer these questions, I presented the following three studies using an implicit atomistic model and performed Langevin dynamics simulations coupled with targeted molecular dynamics (TMD): (1) non-conserved allosteric cycles featured Clp nanomachine mediated remodeling mechanisms of three knotted SPs with 3.1, 5.2 and 6.1-knot types. These simulations reveal knot sliding along the contour of the knotted protein traversed through a rugged conformational landscape. Translocation hindrance of knotted SPs results from the synergetic coordination between knotted topologies and non-native contacts. In contrast to homopolymers, transmission of tension along the peptide chain occurs very differently. Disruption or formation of divergent type contacts along with backbone-backbone contacts can create multiple unfolding pathways with distinct energy barriers. (2) elucidate the effect of mechanical anisotropy on unfolding and translocation of stable substrate proteins with similar global stability but distinct local environments by Clp ATPases. Here, I consider stable DHFR and its two types of circular permutations (CPs), i.e., CP P25 and CP K38, as the SP. The resulting unfolding pathways reveal the unfolding energy barrier is largely determined by the force directionality. Clp nanomachine permits different unfolding mechanisms to regulate the unfolding energy barriers and features different unfolding pathways. Detailed analysis reveals the translocation hindrances arise from a combined effect of formation or disruption of non-native contacts, force directionality, and the arrangement of the primary sequence upon the unfolding. (3) This study focuses on the remodeling mechanism of bulky HaloTag protein using Clp Nanomachine with restrained or unrestrained geometries, which mimics the single-molecule experiments and in-vivo setups respectively. Regardless of unfolding direction or geometry, all unfolding pathways produce significant amounts of non-native contacts that later hinder the translocation mechanisms of HaloTag. In C-N directional unfolding process of HaloTag produces one stable intermediate state. In unrestrained geometry, nanomachine is not able to destabilize this intermediate state, but in restrained geometry, this stable intermediate unravels with the assistance of constant opposing force. Overall, these simulations shed light on mechanistic details of protein unfolding and translocation mediated by AAA+ ATPase nanomachines.

This work was designed to explore the impact of polymer conjugation on cellulase activity and stability. Though cellulase activity simply measures the enzyme's rate of hydrolysis of an anionic, water-soluble cellulose derivative, stability is a bit more nuanced. Stability can refer to an enzyme's thermal, chemical, functional, or thermodynamic stability. To address this, we also created a method to quickly and simply extract thermodynamic parameters of protein unfolding and quantify organic solvent harshness. This dissertation shows the importance of polymer composition, polymer-substrate compatibility, polymer length, and conjugation strategy when rationally designing protein-polymer conjugations to tune protein activity and stability. The resulting FnCel5a-polymer bioconjugates showed increased enzymatic activity due to hydrogen bonding and electrostatic interactions between hydrophilic, cationic polymers and hydrophilic, anionic substrate. In addition to this, our work has demonstrated that polymer dissociation from site-specific FnCel5a-polymer bioconjugates resulted in a loss of enzymatic enhancements gained by conjugation. To mitigate this, we created a hydrolytically stable method of site-specific polymer conjugation. Though polymer attachment site has no significant impact on enzymatic activity, increasing polymer molecular weight enhances the impact of the polymer on enzymatic activity. Generally, polymer conjugation has no significant impact on cellulase activity. However, thermal stability is significantly reduced when large molecular weight incompatible polymers at attached to FnCel5a. The results from this dissertation can be utilized to rationally design protein-polymer bioconjugates in the future, based on the trends and insights gained from these studies.

Cellular protein quality control comprises a network of chaperones that maintain the proteome viability by performing key cellular tasks such as degrading or remodeling misfolded proteins. Bacterial Caseinolytic proteases (Clp) which are responsible for protein degradation include powerful ring-shaped AAA+ (ATPases Associated with diverse cellular Activities) motors with a central narrow pore that unfold and translocate tagged abnormal proteins. Clp ATPase machines thread substrate proteins (SPs) through their central channel by using repetitive ATP-driven subunit motions coupled with axial mechanical forces exerted onto the SP. The molecular details of unfolding and translocation of SPs by these biomolecules are not fully yet understood.Here I present three studies regarding this issue:(1) I perform Langevin dynamics simulations using a coarse-grained model of SPs, Titin I27 and its V13P variant, threading through the ClpY pore. I probe the effect of ClpY surface heterogeneity and changes in pore width on the SP orientation and the direction of applied force during SP unfolding. I contrast mechanisms of SP unfolding in a restrained geometry, as in single-molecule force spectroscopy experiments, and in an unrestrained geometry, as in the in vivo degradation process. In open pore configurations, unfolding of the unrestrained SPs occurs via an unzipping mechanism which involves force application along a weak mechanical direction. In the partially closed pore, unfolding occurs via a shearing mechanism with force application in a strong mechanical direction. By contrast, unfolding of the restrained I27 is limited to a shearing mechanism due to application of force along the strong mechanical direction. I propose that Clp nanomachine plasticity underlies direction-dependent pulling mechanisms that enableversatile SP remodeling actions. (2) I use an implicit solvent model of ClpY and different I27 tandem constructs as SPs and perform Langevin dynamics simulations of cyclical opening and closing of ClpY to mimic the single molecule experiments, in which the N-terminal of I27 and (I27)_4 are restrained and compare and contrast it with in vivo ClpY action setups in which unrestrained I27, (I27)2 and (I27)4 are used as substrates. I find that ClpY unfolds and translocates unrestrained single domain I27 with higher efficiency compared with other cases. Restraining the N-terminal of SP or the presence of external domains significantly reduces the unfolding yield by restraining the orientation of SP. I propose that Clp ATPase biological nanomachines utilize direction depend pulling mechanisms to unfold their substrates.(3) I perform further simulations of unfolding and translocation of knotted proteins through the central pore of ClpY by using an implicit solvent model of ClpY and three knotted proteins with 3_1, 5_2 and 6_1 knot types. I find that unfolding and translocation process results in knot displacement toward the N-terminal and knot tightening caused by non-native contacts hinders the translocation process.

Molecular motors transduce chemical energy –usually from ATP hydrolysis– into directed motion and mechanical work, which is used to perform key functions in almost every cellular process. Molecular motors are particularly important in the maintenance of cellular proteostasis, i.e. the equilibrium between protein synthesis and degradation. ATP-dependent proteases of the AAA+ family, such as ClpXP from Escherichia coli and the eukaryotic 26S proteasome, play a central role in protein degradation. Given its extensive biochemical and structural characterization, ClpXP is a paradigm for the study of the operating principles of eukaryotic and prokaryotic protease machines of the AAA+ family. However, the molecular mechanism by which ClpXP couples the energy from ATP hydrolysis into mechanical work is still poorly understood. Here we used biochemical and single-molecule assays with optical tweezers to directly probe the operation of ClpXP as it unfolds and translocates its protein substrate.Chapter one provides an introduction into the structure and function of ClpXP, which is considered an archetype for the study of AAA+ proteases. This chapter also introduces key concepts about single molecule studies assays with optical tweezers.Chapter two focuses on the study of the mechanisms of force generation and intersubunit coordination of ClpXP. We establish that ClpXP translocates its substrate by using cycles of alternating dwell/burst phases. Phosphate release is the force generating step in the ATP cycle, and ClpXP translocates its substrate in bursts resulting from highly coordinated conformational changes in two to four ATPase subunits. Based on our results, we propose a model where ClpXP must use its maximum firing capacity of four subunits to unfold stable substrates like GFP. Interestingly, the average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying a constant translocation cycle governed by an unidentified “internal clock”. Together, our results indicate that ClpXP operates with constant “rpm” but uses different “gears”.Chapter three describes the complete mechanochemical cycle of ClpXP, as well as the coupling and power efficiency of the motor. We show that ADP release and ATP binding happen non-sequentially during the dwell, while ATP hydrolysis and phosphate release occur during the burst. ADP release is the rate-limiting transition of the ATP cycle. Interestingly, the size of the highly-conserved translocating loops within the ClpX pore has been evolutionarily optimized to maximize motor power generation, as well as the coupling between chemical and mechanical cycles of the motor. Finally, we presente evidence showing that the conformational resetting of these loops between consecutive bursts seems to determine ADP release from individual ATPase subunits and the overall duration of the motor’s cycle, which explains the observed “internal-clock mechanism” of ClpXP.

Protein homeostasis is crucial for a cell's viability and therefore an organism's survival. Proteins, upon translation, must reach a three-dimensional structure known as their native state. This structure defines a particular protein’s function from a set of diverse enzymatic activities. Proteins must traverse a rough, many multi-dimensional energy landscape in order to approach their native state at the native basin of attraction. While some small, single-domain proteins are able to fold spontaneously and directly to their native state, some larger and multi-domain proteins, fold via multiple parallel pathways, at times engaging with competing basins of attraction, existing as an ensemble of misfolded structures known as non-native states. Proteins that become stuck in non-native states are prone to forming aggregates, due to exposed hydrophobes. Such aggregates have been implicated in various neurological diseases such as Parkinson's and Alzheimer's. Nature has evolved a protein quality control system which employs biological nanomachines, working to moderate off-pathway reactions, thereby modulating a balance between native and non-native proteins. There are two components to this system: protein degradation and protein folding assistance. While various components of this system engage in sundry tasks, I have focused on a unique system from each subsystem: p97, having unfolding activity and GroEL, having protein-folding assistance activity. p97, a ubiquitous hexameric, dual-ring ATPase found in eukaryotes, utilizes its high ATPase-activity D2 domains to drive dramatic allosteric conformational changes between distinct nucleotide-bound states. This results in paddling of highly-conserved loops found in the interior of the ring to pull proteins through its narrow central pore by repetitive engagement and release. Translocation of a substrate protein through this pore leads to unfolding, in preparation for proteolysis. I have used normal mode analysis to investigate how the collective motions of the p97 monomer, dimer and hexamer underlie p97's allosteric transitions. p97's open conformation shows common behavior in the two most-dominant collective motions in each system. Namely, a clockwise torsional motion suitable for directionality of substrate handling and an axial swing-like motion that transmits force to the substrate. Additionally, I looked at the directional correlation of the collective motions, which reveals a sequential nature of p97 and further suggests CW directionality in the dimer. Further, I performed a structural perturbation method, probing each residue’s energetic contribution. Coupling this to the aforementioned results, along with details of the collective motions of the systems, reveals a network of 23 residues which are important to the allosteric signaling network of p97. The chaperonins, providing protein-folding assistance via forced unfolding and sequestration of substrates, comprise two groups with distinct kinetics: the bacterial Group I utilize concerted allosteric motions, while the eukaryotic Group II utilizes sequential. I investigated protein folding coupled to the sequential allostery of a mutant bacterial GroEL using Langevin dynamics and a de novo candidate for folding assistance with a coarse-grained model. The sequential mutant shows dramatically reduced encapsulation ability. The confined substrate unfolding outcome is unchanged from wild-type. These data suggest encapsulation may not be a requirement for sequential allosteric protein folding assistance.

Mass spectrometry (MS) serves as an indispensable technology for modern pharmaceutical drug discovery and development processes, where it is used to assess ligand binding to target proteins and to search for biomarkers that can be used to gauge disease progression and drug action. However, MS is rarely treated as a screening technology for the structural consequences of drug binding. Instead, more time-consuming technologies capable of projecting atomic models of protein-drug interactions are utilized. In this thesis, ion mobility-mass spectrometry (IM-MS) methods are developed in order to fill these technology gaps. Principle among these is collision induced unfolding (CIU), which leverages the ability of IM to separate ions according to their size and charge, in order to fingerprint gas-phase unfolding pathways for non-covalent protein complexes. Following a comprehensive introductory chapter, we demonstrate the consequences of sugar binding on the CIU of Concanavalin A (Con A) in Chapter 2. Our CIU assay reveals cooperative stabilization upon small molecule binding, and such effect cannot be easily detected by solution phase assays, or by MS alone. In Chapter 3, the underlying mechanism of multi-protein unfolding is systematically investigated by IM-MS and molecular modeling approaches. Our results show a strong positive correlation between monomeric Coulombic unfolding and the tetrameric CIU process. This provides strong evidence that multi-protein unfolding events are initiated primarily by charge migration from the complex to a single monomer. In Chapter 4, the interactions between human histone deacetylase 8 (HDAC8) and poly-r(C)-binding protein 1 (PCBP1) are investigated by IM-MS. Our data suggest that these proteins interact with each other in a specific manner, a fact revealed by our optimized ESI-MS workflow for quantifying binding affinity (KD) for weakly-associated hetero-protein complexes. In Chapter 5, the translocator protein (TSPO) dimer from Rhodobacter sphaeroides, as well as its disease-associated variant forms, is analyzed by IM-MS and CIU assays. By utilizing a combination of CIU and collision induced dissociation (CID) stability data, an unknown endogenous ligand bound to TSPO is detected and identified.

Proteins are large complex molecules that play important roles in cellular activities such as DNA replication, molecular transport, cell immunity, and regulatory activities. Based on these roles, the half–lives of proteins within cells vary widely, but when the time comes they need to be degraded regardless.To address this issue, cell utilizes a protein quality control (PQC) system that unfolds and degrades proteins. One of the pathways used by PQC is selective degradation via molecular chaperones. The focus of this study is on the molecular chaperones that assist in unfolding and the mechanism through which they interact with their substrate proteins (SP). Unfolding chaperones are members of the AAA+ (ATPases Associated with various cellular Activities) family that form oligomers, typically hexamers, that enclose a central pore. It has been suggested that the conserved hydrophobic loop located at the entrance to the pore engages the SP and remodels it by exerting mechanical forces via a paddling motion. However the mechanism of SP processing is not known yet. Here I am presenting four studies regarding this issue: (1) Mechanism of transient binding and release of the SP during allosteric cycle of p97 (a group II AAA+ machine). (2) Mechanism of SP unfolding and translocation by p97. (3) Asymmetric processing of SP protein in sequential allosteric cycles. (4) The role of I–domain in unfolding and translocation of SulA by ClpY (group I) nanomachine. I use molecular dynamics simulations to probe the interaction of p97 and a long extended peptide as the SP. Mechanical pulling of the substrate through the p97 pore reveals that less work is required for translocation from the D1 (ring I) towards the D2 (ring II) compartment than in the opposite direction. Also based on simulations of SP binding to p97, I found that transient binding inside the pore happens through a lever mechanism in which SP binding to Glu554 supersede Arg599 as the pore closes during the allostric cycle. I perform coarse–grained (one bead per amino acid) Langevin dynamics simulations of a tagged four–helix bundle protein introduced to p97. The results indicate that interaction with both binding site inside D2 pore (Arg599), and the hydrophobic loop of D2 (Trp551, Phe551), is required for unfolding and translocation of the SP. I conduct further simulations of the same system with both clockwise and counterclockwise directionality of sequential intra–ring allosteric motion. I find that in both directions the SP conformational changes take place along the same pathway. However, the rates and yields of SP unfolding and translocation are higher in the clockwise direction due to better SP handling between adjacent subunits. Finally I introduced an improved coarsed–grained model (two bead per amino acid) parametrized using a statistical potential. I carried out Langevin dynamics simulations of the ClpY nanomachine and its natural substrate SulA. The results show that the absence of Clpy I–domain induces a deficiency in SP unfolding and therefore affects translocation. In this case, the I–domain binds SulA and facilitates the unfolding process by exerting additional forces on SP.

Protein mechanical responses play a critical role in a wide variety of biological phenomena, impacting events as diverse as muscle contraction and stem cell differentiation. Recent advances in both experimental and computational techniques have provided the opportunity to explore protein constitutive properties at the molecular level. However, despite these advances many questions remain about how proteins respond to applied mechanical forces, particularly as a function of load magnitude. In order to address these questions, relatively simple helical structures were computationally tested to determine the mechanisms and kinetics of unfolding at a range of physiologically relevant load magnitudes. Atomically detailed constant force molecular dynamics simulations combined with the Milestoning kinetic analysis framework revealed that the mean first passage time (MFPT) of the initiation of unfolding of long (~16nm) isolated helical domains was a non-monotonic function of the magnitude of applied tensile load. The unfolding kinetics followed a profile ranging from 2.5ns (0pN) to a peak of 3.75ns (20pN) with a decreasing MFPT beyond 40pN reflected by an MFPT of 1ns for 100pN. The application of the Milestoning framework with a coarse-grained network analysis approach revealed that intermediate loads (15pN-25pN) retarded unfolding by opening additional, slower unfolding pathways through non-native [pi]-helical conformations. Analysis of coiled-coil helical pairs revealed that the presence of the second neighboring helix delayed unfolding initiation by a factor of 20, with calculated MFPTs ranging from 55ns (0pN) to 85ns (25pN per helix) to 20ns (100pN per helix). The stability of the coiled-coil domains relative to the isolated helix was shown to reflect a decreased propensity to break flexibility restraining intra-helix hydrogen bonds, thereby delaying [psi] backbone dihedral angle rotation and unfolding. These results show for the first time a statistically determined profile of unfolding kinetics for an atomically detailed protein that is non-monotonic with respect to load caused by a change in the unfolding mechanism with load. Together, the methods introduced for analyzing the mechanical response of proteins as well as the timescales determined for the initiation of unfolding provide a framework for the determination of the constitutive properties of proteins and non-biological polymers with more complicated geometries.

Proteins are usually thought of as having a chemical role in cells (i.e. enzymes), however many of them have structural roles as well. These are termed mechanochemical proteins, as they perform both chemical and mechanical functions. The focus of this work will be on the mechanical behavior of three types of proteins involved in mechanotransduction namely, microtubules (MTs), actin, and the titin-telethonin complex. Both MTs and actin filaments are cytoskeletal filaments, which work to maintain the shape and size of the cell. MTs serve as tracks for molecular motors and both align and separate the chromosomes during cell division. The actin filament, in addition to defining the edges of cells, binds to myosin to create the forces required for muscle contractions. The titin-telethonin complex, involved in mechanotransduction in muscles, serves as an anchor for tandems of immunoglobulin domains in muscle cells. For all of these proteins, their response to applied forces is crucial to their biological function in the cell. It is the goal of this work to determine the mechanical properties of these systems, that underlie their cellular functions, using multiscale simulations.

Protein quality control is critical in maintaining cell viability. Recognitionand degradation of aberrant proteins in the cellular environment is essential,as many neurodegenerative diseases are linked to protein misfolding andaggregation. Powerful AAA+ ATPases, such as ClpY and p97, play avital role in the degradation pathway of PQC. These nanomachines form hexameric macromolecularassemblies which selectively recognize and thread proteins tagged fordegradation through their narrow central pore to be delivered to sequesteredprotease components for degradation. Flexible loops with aconserved G-hydrophobic-aromatic-G sequence reside within the central pore andundergo a large scale paddling motion resulting from ATPhydrolysis. The loops impart mechanical forces onto substrateproteins (SP) which results in unfolding and translocation. While the overallmechanism of these macromolecular machines isgenerally understood, molecular details of SP processing have not been established. The focus of this study is on the molecular details of SPprocessing by the single ring ClpY ATPase, whichhas been well characterized crystallographically in multiple nucleotide boundstates. Here, I am presenting studies based on four aspects regarding this problem : (1) Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allostericcycles of the ClpY ATPase, (2) Asymmetric processing of a substrate protein insequential allosteric cycles of the ClpY nanomachine, (3) Thedependence of SP topology on unfolding and translocation mechanisms by theClpY nanomachine, and (4) Structure mediated unfolding and translocationpathways of SulA by pulling through non-allosteric AAA+ pores. I used coarse-grained Langevindynamics simulations to probe the unfolding and translocation of a four-helix bundlemodel SP using the allosteric transitions of ClpY. My results indicate thatunfolding occurs by unraveling from the SP's tagged C-terminus, resulting in athree helix bundle. This minimally unfolded, obligatory unfolding intermediate,is competent for translocation. Translocation occurs through sharpstepped transitions indicating a powerstroke mechanism. To further understandthe coupling between intra-ring motions of ClpY and mechanical action applied tothe SP, I performed additional simulations of clockwise,counterclockwise and random allosteric mechanisms. Myresults indicate that the SP unfolding and translocation mechanism is independent of thedirectional allosteric mechanism. The work provided by single SP-loopinteractions allows the SP to sample identical conformational states; However,the rates and yields are affected, with clockwise allostery emerging as the mostefficacious due to the favorable torque applied onto the SP. Next, I have probedthe dependence of SP topology by examining ClpY processing of a SP withalpha/beta topology. Comparison with the all-alpha SP indicate a wellpreserved unfolding event by unraveling at the C-terminus and translocationvia a powerstroke mechanism. The effective forces for unfolding andthe kinetics depend on topology. Last, I have probed theunfolding and translocation of a natural SP of ClpY, SulA, by performingminimalistic simulations pulling through non-allosteric AAA+ pores. The results indicatethat initial unfolding near the C-terminus is required for initiation oftranslocation. Interactions with the surface of the pore facilitate unfoldingand translocation by decreasing the energy barriers.

The intricate functions of membrane proteins would not be possible without bends or breaks that are remarkably common in transmembrane helices. The frequent distortions are nevertheless surprising because backbone hydrogen bonds should be strong in an apolar environment, potentially rigidifying helices. It is therefore mysterious how distortions can be generated by evolutions. Through my studies on bacteriorhodopsin and Ca2+-ATPase, I found that helix distortions are facilitated by shifting hydrogen bonding partners. My results explained how evolution has been able to liberally exploit transmembrane helix bending by for optimizing membrane protein structure, function and dynamics.It has recently been found that the stability of bacteriorhodopsin assessed through unfolding did not achieve equilibrium. Here I made a new equilibrium test by measuring the transition of folded bacteriorhodopsin to unfolded bacterioopsin. My result suggests that the energetic effects of most mutations that have been studied before may be focused on the folded state. To further investigate how hydrogen bonds may shape transmembrane helices, I sought a non-perturbing method to measure strengths of both backbone and side-chain hydrogen bond strengths, because backbone hydrogen bonds cannot be probed by mutation and mutagenesis to remove side-chains may not mimic the breaking of side-chain hydrogen bonds. I therefore decided to employ the equilibrium hydrogen / deuterium fractionation factors (phi-value) for hydrogen bonds. I used model compounds to study the relationship between phi-value and the hydrogen bond free energy. By applying this relationship, I found that hydrogen bonding stabilizes enzyme intermediate state by up to 4 kcal/mol more than the resting state and marginally favor soluble-protein unfolding.To measure hydrogen bond strengths in a membrane protein, I focused on the isolated voltage-sensor domain of the voltage-dependent potassium-selective channel. By utilizing its 2D NMR spectrum, I obtained phi-values and thus free energies for 70 % of the backbone hydrogen bonds in this membrane protein. I found that it has similar backbone hydrogen bond strength to water-soluble proteins on average. Moreover, I found that the flexible (dynamic) point of a transmembrane helix in this protein can be predicted from the backbone hydrogen bond strengths in that helix.

Computer simulations on the molecular dynamics of biological molecules can be used to explore the behavior of large molecules with a level of detail not possible in experiments. It is necessary to be cautious, however, when designing and interpreting such simulations, as the techniques commonly used to improve efficiency of simulations can lead to unrealistic results. The work presented in this dissertation explores ways in which the accuracy of molecular dynamics simulations can be both improved and validated by experimental data, primarily through the use of potentials of mean force (PMFs). Experimental input was used in the development of an umbrella sampling protocol by fitting restraining potentials to an experimental PMF. The method was tested on a model peptide using a ”guiding” PMF from simulations and then validated using an experimental PMF from force manipulation studies on a mechanical protein. The results show that the experimentally guided umbrella sampling replicates the appropriate pathways for both systems, whereas naively chosen potentials fail to do so. Experimental findings were also used in the design of steered molecular dynamics simulations on the β domain of streptokinase. High-temperature simulations were used to smooth the energy surface and enable the system to explore alternate unfolding pathways. The results show three distinct pathways, in agreement with experimental evidence of three types of behavior under force. The simulations reveal that the source of the differences are hydrophobic interaction in the core of the protein. Multi-dimensional PMFs were calculated to describe these pathways energetically. All-atom simulations were also used to study a different type of system, the interactions between DNA and a Polyamidoamine dendrimer. Both the dendrimer and DNA were found to deform substantially upon binding. While the interactions were shown to be driven primarily by electrostatics, we also find that ordered waters extend the interaction distance beyond the range of direct electrostatics for one orientation of the dendrimer. These water effects contribute almost a third of the total interaction free energy of the system. PMFs calculated in these simulations were used to calculate force extension curves which agree with experiments on DNA condensed by dendrimers.

Modification of blood solutes with methoxy-polyethylene glycol (PEGylation) increases their size and half life in vivo. With three PEG reagents, we have created several species of PEGylated human serum albumins and examined their properties in vitro. Determination of size was undertaken with gel filtration, SDS-polyacrylamide gel electrophoresis, and surface enhanced laser desorption/ionization time of flight (SELDITOF)mass spectrometry. All demonstrated a significant increase in the size of the PEGylated albumin species, which was reflected in significant nonideal behavior during colloid osmotic pressure (COP) studies. Urea-induced protein unfolding experiments demonstrated that PEGylation does not significantly alter the stability of albumin’s fluorophore, tryptophanyl residue 214 (Trp 214). Solute-quenching studies of all but the most heavily modified albumin species monstrated no significant change in the fluorescent properties Trp 214. We believe PEGylated albumins have promise as resuscitative agents for hypoperfusion secondary to shock.