Simulation vs. Reality: A Comparison of In Silico Distance Predictions with DEER and FRET Measurements

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by Förster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions

Proteins on the edge : transitions of structure ensembles in protein unfolding and protein-protein binding

Proteins move. Their incessant fluctuations are governed by a complex interplay between thousands of atoms. Experimental structures, providing exact coordinates for every atom, hence only represent the average of a diverse ensemble of interchanging conformations. Molecular motion is often the barely understood link between structure and biological function. The present work examines two different processes that put proteins on the edge of moving from one global state to another. At the moment of transition, perturbation or, indeed, biological action, benign structure fluctuations can, it seems, turn into major forces. Chains of spectrin repeats apparently rely on structure flexibility to achieve a smooth response to external force. Single molecule atomic force microscopy experiments on this domain, in accord with simulations, showed clear traces of structure fluctuation. On the verge of disruption, thermal fluctuations decide how much extension a spectrin repeat tolerates and whether or not unfolding is blocked by intermediate non-native structures. This picture was supported by experiments and simulations on mutated repeats. The elasticity of the membrane skeleton and, for example, red blood cells, may thus to some extent depend on chaotic motions within single protein domains. Structure fluctuations also affect the process of protein-protein interaction, but the interplay of protein flexibility and recognition remains far from understood. I performed and compared molecular dynamics simulations on 17 protein complexes as well as their free components. Free interaction patches turned out more flexible than the remaining protein surface. However, contrary to common sense, binding does not generally restrict protein flexibility and conformational entropy may be lost but also gained in the process. Current models of recognition do not account for overall protein flexibility or make assumptions that are incompatible with kinetic observations. I combined the simulation data with systematic docking calculations and derived a new model for this process. Often, only subsets of the two free structure ensembles were mutually compatible. A conformer selection step may thus impede the rate of recognition. Protein fluctuations seem to be actively involved in the binding reaction and influence or even control the speed of recognition as well as the stability of the complex

ECORI ENDONUCLEASE-DNA COMPLEXES STUDIED BY THERMODYNAMICS AND ELECTRON SPIN RESONANCE SPECTROSCOPY

This work focuses on adducing general principles applicable to site-specific protein-DNA interactions by linking function to structural, thermodynamic and dynamic properties. The interaction of EcoRI endonuclease with specific, miscognate, and nonspecific DNA sequences is used as a model for protein-DNA interactions. We use four pulse Double Electron-Electron Resonance (DEER) Electron Spin Resonance (ESR) experiments to map distances and distance distributions between nitroxide spin labels placed at positions within the ‘arms' and the main domain of the EcoRI homodimer. These experiments show that the DNA occupies a similar binding cleft and is enfolded by the arms of the enzyme in all three classes of EcoRI-DNA complex. Additionally, changes in dynamics of main domain and arm residues within the three complexes were explored using Continuous Wave (CW) ESR spectroscopy. A position adjacent to a protein-phosphate contact shows decreased mobility relative to other arm residues that are not at the protein-DNA interface. Signal from this position shows the largest amount of an immobile component in the specific complex, progressively less immobile in the miscognate and nonspecific complexes. This fits with distribution breadths from DEER-ESR spectra and biochemical evidence that the nearby phosphate contact is made only in the specific complex. Residues at other positions show mobilities that are in agreement with our hypothesis that residues in the arms would be relatively more mobile than those in the main domain Using Electron Spin Echo Envelope Modulation (ESEEM) ESR we show that the paramagnetic Cu2+ ion is coordinated by an imidazole nitrogen. These experiments thus reveal a novel metal ion binding site. DEER measurements of distances between Cu2+ ions and Cu2+-nitroxide distances in the homodimeric EcoRI-DNA complex establish that the Cu2-coordinating residue is histidine 114, which is proximal to but not at the active site. This is consistent with our biochemical studies that show that Cu2+ cannot replace Mg2+ as a catalytic cofactor but instead completely inhibits EcoRI cleavage. We also use isothermal titration calorimetry (ITC) to directly determine a stoichiometry of two Cu2+ ions bound per homodimeric EcoRI-DNA complex; that is, each histidine 114 coordinates one Cu2+ ion

Structural folding dynamics of an archetypal conformational disease using Nuclear Magnetic Resonance Spectroscopy

Members of the serpin (serine protease inhibitor) superfamily of proteins regulate key physiological processes through their ability to undergo major conformational transitions. In conformational diseases, native protein conformers convert to pathological species that polymerise. Structural characterization of these key transitions is challenging. Mechanistic intermediates are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. I use Nuclear Magnetic Resonance (NMR), and Circular Dichroism (CD) spectroscopy, to investigate the interrelated processes of serpin folding, misfolding and polymerisation in solution using the 45kDa prototypic serpin á1-antitrypsin, the recent assignment of the backbone resonances of á1-antitrypsin by our group, allows us to ask more sophisticated questions by a range of NMR techniques to study its structure and dynamics. In this study, I analysed early unfolding behaviour of á1-antitrypsin across a urea titration within what is apparently the largest two-states system yet characterised. In order to assess the dynamics of the native state, I have used hydrogen/deuterium exchange nuclear magnetic resonance spectroscopy (HDXNMR) to characterise motions on the slow (ms) timescale. I have conducted a detailed analysis of residue-specific changes in protection from exchange across a pH titration using SOFAST-HMQC. This is complemented by a detailed a preliminary analysis of fast motions (ps-ns) using NMR relaxation experiments. Moreover, a forme fruste deficiency variant of á1-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo was characterized in this study. Lys154Asn á1-antitrypsin populates an intermediate ensemble along the polymerisation pathway at physiological temperatures. Together, this study shows how the use of powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions, provides novel insights into pathological conformational behaviour

Pseudomonas aeruginosa PA3859: From Structure to Function

The aim of this work has been the structural and functional study of a putative carboxylesterase purified from P. aeruginosa, namely PA3859. The protein has been purified from the wild type and a preliminary biochemical charcterization was carried out. The PA3859 gene was then cloned and the recombinant protein was expressed in E. coli (Chapter 2). The recombinant PA3859 was successfully crystallized and its 3D crystal structure was determined (Chapter 3 and 4). Starting from the enzyme 3D structure, an approach involving in silico, in vitro and in vivo assays lead to the reliable determination of the PA3859 physiological function (Chapter 5, 6, 7)

Strategies to improve the immunogenicity of subunit vaccine candidates

Subunit vaccines contain highly defined macromolecular components of a pathogen that are capable of eliciting protective immunity. They possess several advantages over other vaccine types (e.g. live attenuated and inactivated) such as improved safety profiles, highly defined nature, ease of production, and potential for lower cost of goods. One critical limitation of subunit vaccines, however, is their weak immunogenicity owing to their inability to replicate, monovalent structures, and the absence of other immunostimulatory components. Common approaches to enhance the immunogenicity of subunit vaccines include polyvalent antigen display strategies, the use of adjuvants, etc. The polyvalent antigen display strategy requires the use of a scaffold, which can be protein-based or some other materials. Chapter 2 focuses on the biophysical properties of a potential scaffold for polyvalent antigen display-Bacillus anthracis lumazine synthase (LS), an icosahedral homo-oligomeric protein. LS in PBS buffer showed a minor thermal transition around 50 ᵒC, and a major one at 95 ᵒC. The minor transition arose from the dissociation of the LS/phosphate complex, which formed in PBS buffer at room temperature. The major transition corresponded to the dissociation of LS oligomers, thermal unfolding, and aggregation. In chapter 3, I describe an attempt to develop ricin vaccine candidates in which LS was used as a scaffold to achieve polyvalent display of a linear neutralizing epitope (designated PB10) from ricin. PB10 was genetically inserted onto the C terminus of LS, and the fusion protein (designated LS_PB10) was expressed in an E.coli system. LS_PB10 self-assembled into spherical particles. Fusion of the PB10 peptide did not affect the structure and stability of LS. LS_PB10 showed tight binding to a mAb targeting the PB10 epitope. Immunization of LS_PB10 in mice elicited a moderate level of anti-ricin serum titers, which, however, failed to offer protection during a challenge study using a 10x lethal dose of ricin. Such an unsatisfactory end result may be attributable to 1) limited efficacies of using the PB10 epitope alone, 2) loss of secondary structure of PB10 on LS; 3) an epitope suppression effect induced by the highly immunogenic nature of the LS scaffold. Studying antigen/adjuvant compatibility is critical for the development of adjuvanted vaccine formulations. Chapter 4 discusses the utilization of biophysical tools to understand effects of two emulsion-based adjuvants (designated as ME.0 and SE) on the structure and thermal stability of alpha-toxin (AT), a potential vaccine candidate for Staphylococcus aureus infection. Both adjuvants are oil-in-water (O/W) emulsions using squalene as the oil phase. DSC analysis showed the ME.0 emulsion thermally destabilized AT, probably because of changes in the buffer composition of AT upon mixing. The SE emulsion caused increased alpha-helix and decreased beta-sheet content in AT, and a blue shift in Trp fluorescence emission spectra of AT. DSC analysis showed SE exerted a dramatic thermal stabilization effect on AT, probably attributable to an interaction between AT and SE. Size exclusion chromatography showed a complete loss in the recovery of AT when mixed with SE, but not ME.0, indicating a high degree of interaction with SE. The goal of protein formulation development is to identify optimal conditions for long-term storage. Certain commercial conditions (e.g., high protein concentration or turbid adjuvanted samples) impart additional challenges to biophysical characterization. Formulation screening studies for such conditions are usually performed using a simplified format in which the target protein is studied at a low concentration in a clear solution. The failure of study conditions to model the actual formulation environment may cause a loss of ability to identify the optimal conditions for target proteins in their final commercial formulations. In chapter 5, we utilized a steady-state/lifetime fluorescence-based high-throughput platform to develop a general workflow for direct formulation optimization under analytically challenging but commercially relevant conditions. A high-concentration monoclonal antibody and an Alhydrogel-adjuvanted antigen were investigated. A large discrepancy in screening results was observed for both proteins under these two different conditions (simplified versus commercially relevant). This study demonstrates the feasibility of using a steady-state/lifetime fluorescence plate reader for direct optimization of challenging formulation conditions and highlights the importance of performing formulation optimization under commercially relevant conditions

Understanding and optimizing the interactions of functional species in mesostructured materials with diverse transport properties

Mesostructured inorganic-organic hybrid materials have high interfacial areas, through-connecting mesoscale (2-50 nm) channels, and high mechanical and chemical robustnesses that can be exploited for diverse technological applications, especially those that involve the transport of charges, ions or molecules. These materials can, moreover, accommodate a broad assortment of functional molecular species that can mediate or instill transport properties. One interesting example is membrane proteins, which are biomacromolecules that transport ions or molecules across biological lipid bilayers at high rates and selectivities; for instance, the membrane protein proteorhodopsin actively transports H+-ions in response to light, which might be harnessed for solar-to-electrochemical energy conversion. Mesostructured inorganic-organic hybrid materials that include macroscopically aligned proteorhodopsin species are expected to generate bulk ion gradients across host materials under illumination. Here, a solution-based synthetic protocol is presented that allows high concentrations (up to 15 wt%) of active proteorhodopsin species to be incorporated within mesostructured silica membrane hosts. Synthesis conditions and compositions were selected to stabilize proteorhodopsin molecules in the presence of the structure-directing surfactant and soluble network-forming silica species that co-assemble to form mesostructured silica host matrices, as established by small-angle X-ray diffraction analyses. Multidimensional solid-state NMR spectra show that proteorhodopsin molecules incorporated within mesostructured silica hosts retain native-like structures, though with some interesting differences. The optical absorbance behaviors of proteorhodopsin guests in the synthetic mesostructured silica hosts correspond to the photochemical reaction cycles of proteorhodopsin in near-native environments that are associated with the active transport of H+-ions. The collective synthetic, structural and functional understanding developed here is leveraged to synthesize durable and optically transparent self-supporting mesostructured silica films that include high loadings of proteorhodopsin, as well as other membrane proteins. In separate mesostructured material systems, functional guest species at the mesostructure interfaces are shown to dramatically influence bulk material performances. Specifically, aminoalkyl-fullerene functionalities at the mesochannel surfaces of mesoporous silica can substantially modify the sorption and release behaviors of pharmaceutical species from these materials, while interfacial alkyl functionalities in polymer:fullerene heterojunctions can promote efficient separation of photo-generated charges

Thermodynamics and Kinetics of Iso-1-cytochrome c Denatured State

Various diseases result from protein misfolding. Curing these conditions requires understanding the principles governing folding. Efforts toward understanding how proteins fold have focused on the transition state rather than the earliest folding events. We study these initial events using the assumption that protein folding must involve the formation of the most primitive structure possible – a simple loop. Our laboratory has developed a system of studying simple loops in the denatured state using c-type cytochromes. New insights into how the properties of these loops impact the denatured state are outlined in this thesis. First, studies on a 22-residue loop revealed a previously unreported finding that equilibrium loop formation was not strongly affected by sequence composition. While loop formation rates depended only on sequence composition, loop breakage rates also depended on sequence order. Second, thermodynamic and kinetic studies on homopolymeric inserts in “poor” and “good” solvents revealed that homopolymeric non-foldable protein sequences behave like a random coil. However, heteropolymeric foldable sequences have scaling factors higher than those of a random coil, suggesting the presence of residual structure in denatured proteins. Thus, peptide models with homopolymeric sequences do not adequately describe the nature of foldable sequences. Third, we investigated the kinetics of reversible oligomerization in the denatured state using a P25A yeast iso-1-cytochrome c variant. The findings indicated that intermolecular aggregation in a denatured protein is extremely fast – 107-108 M-1s-1 and that the P25A mutation strongly affects intermolecular aggregation. This work suggests that equilibrium control of folding versus aggregation is advantageous for productive protein folding in vivo. Fourth, we use time-resolved FRET to follow compact and extended distributions of a protein under denaturing conditions. Our findings revealed three major populations in the unfolded state when no loop is present whereas only two populations remain when the loop forms. The most extended population is lost upon loop formation showing that simple loop formation dramatically constrains the denatured state. Thus, thermodyamic and kinetic studies on simple loops using a variety of spectroscopic techniques have enhanced understanding of the initial events of protein folding and the role of the denatured state in modulating protein aggregation