Browsing by Author "Oas, Terrence G"

The structural plasticity conferred by conformational flexibility has increasingly been recognized as a likely determinant of function. For example, multiscale heterogeneity in the calmodulin central helix most likely helps it in binding over 100 protein targets, and a concerted motion seen in both nuclear magnetic resonance (NMR) and crystal structures of ubiquitin is proposed to underlie its functional plasticity of promiscuous binding to many different proteins with high affinity. However, flexibility is manifested in a variety of ways, depending both on the protein itself and on how it is observed. Conformational heterogeneity (the term we use for flexibility when studied by X-ray crystallography) is evident in electron density, either as fully separated peaks or as anisotropic density shapes showing fluctuation of atom groupings. Many phenomena contribute to conformational heterogeneity in crystal structures, from diverse crystal contacts to functionally relevant conformational fluctuations on a wide range of time and size scales.

In addition to ubiquitin and calmodulin, the Staphylococcus aureus virulence factor staphylococcal protein A (SpA) is an example of a highly heterogeneous protein. SpA is a major contributor to bacterial evasion of the host immune system, through high-affinity binding to host proteins such as antibodies, von Willebrand factor, and tumor necrosis factor receptor 1 (TNFR1). The protein includes five small three-helix-bundle domains (E-D-A-B-C) separated by conserved flexible linkers. Prior attempts to crystallize individual domains in the absence of a binding partner were apparently unsuccessful. There are also no previous structures of tandem domains. In this thesis, I report the high-resolution crystal structures of a single C domain (collected at both cryogenic and room temperatures), a single A domain, and two B domains connected by the conserved linker. All four apo structures exhibit extensive multiscale conformational heterogeneity, which required novel modeling protocols. Comparison of domain structures shows that helix1 orientation is especially heterogeneous, coordinated with changes in sidechain conformational networks and contacting protein interfaces.

The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure wasn't properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. In this thesis, I report the 2.3-A structure of a fully folded SpA domain in complex with Fc. My structure indicates that there are extensive structural rearrangements necessary for binding Fc, including concerted rotamer changes and coupled backbone rearrangements that lead to a difference in helix1 angle. The conformational heterogeneity of the helix1/2 interface is also eliminated in the complex, with previously poly-rotameric interfacial residues locking into single rotamer conformations. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur in SpA and in its multiple binding partners and may be an important structural paradigm in other functionally plastic proteins.

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.

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.

RNA recognition frequently results in conformational changes that optimize

intermolecular binding. As a consequence, the overall binding affinity of RNA

to its binding partners depends not only on the intermolecular interactions

formed in the bound state, but also on the energy cost associated with changing

the RNA conformational distribution. Measuring these conformational penalties

is however challenging because bound RNA conformations tend to have equilibrium

populations in the absence of the binding partner that fall outside detection by

conventional biophysical methods.

In this work we employ as a model system HIV-1 TAR RNA and its interaction with

the ligand argininamide (ARG), a mimic of TAR’s cognate protein binding partner,

the transactivator Tat. We use NMR chemical shift perturbations (CSP) and NMR

relaxation dispersion (RD) in combination with Bayesian inference to develop a

detailed thermodynamic model of coupled conformational change and ligand

binding. Starting from a comprehensive 12-state model of the equilibrium, we

estimate the energies of six distinct detectable thermodynamic states that are

not accessible by currently available methods.

Our approach identifies a minimum of four RNA intermediates that differ in terms

of the TAR conformation and ARG-occupancy. The dominant bound TAR conformation

features two bound ARG ligands and has an equilibrium population in the absence

of ARG that is below detection limit. Consequently, even though ARG binds to TAR

with an apparent overall weak affinity ($\Kdapp \approx \SI{0.2}{\milli

\Molar}$), it binds the prefolded conformation with a $K_{\ch{d}}$ in the nM

range. Our results show that conformational penalties can be major determinants

of RNA-ligand binding affinity as well as a source of binding cooperativity,

with important implications for a predictive understanding of how RNA is

recognized and for RNA-targeted drug discovery.

Additionally, we describe in detail the development of our approach for fitting

complex ligand binding data to mathematical models using Bayesian

inference. We provide crucial benchmarks and demonstrate the

robustness of our fitting approach with the goal of application

to other systems. This thesis aims to provide new insight into

the dynamics of RNA-ligand recognition as well as provide new

methods that can be applied to achieve this goal.

Electrostatic interactions are of fundamental importance in determining the structure and stability of macromolecules. For example, charge-charge interactions modulate the folding and binding of proteins and influence protein solubility. Electrostatic interactions are highly variable and can be both favorable and unfavorable. The ability to quantify these interactions is challenging but vital to understanding the detailed balance and major roles that they have in different proteins and biological processes. Measuring pKa values of ionizable groups provides a sensitive method for experimentally probing the electrostatic properties of a protein.

pKa values report the free energy of site-specific proton binding and provide a direct means of studying protein folding and pH-dependent stability. Using a combination of NMR, circular dichroism, and fluorescence spectroscopy along with singular value decomposition, we investigated the contributions of electrostatic interactions to the thermodynamic stability and folding of the protein subunit of Bacillus subtilis ribonuclease P, P protein. Taken together, the results suggest that unfavorable electrostatics alone do not account for the fact that P protein is intrinsically unfolded in the absence of ligand because the pKa differences observed between the folded and unfolded state are small. Presumably, multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function.

A clear description of the unfolded state is important for understanding protein folding/misfolding reactions. In addition to general ensemble-averaged properties, distributional residue-specific information is particularly necessary for identifying the molecular causes of many protein misfolding diseases. To this end, an anomalous SAXS (small angle X-ray scattering) technique was developed that provides residue-to-residue distance distribution information for unfolded proteins under physiological conditions. A peptide corresponding in sequence to the first helix of λ repressor was used for preliminary experiments with the proposed technique. Selenium and mercury labels were attached to the termini of the peptide and SAXS data of the labeled peptide were collected at the Argonne National Laboratory. End-to-end distance distribution for selenium-labeled peptide was obtained and the viability of the method was discussed based on experimental and simulation results.

Understanding the interconversion between the thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was used as a model system to elucidate the general folding/unfolding of an intrinsically unstructured protein (IUP) both in the absence and presence of ligands.

P protein was previously characterized as an intrinsically unstructured protein, and it is predominantly unfolded in the absence of ligands. Addition of small anions can induce the protein to fold. Therefore, the folding and binding are tightly coupled. Trimethylamine-N oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic time courses at various final TMAO concentrations showed multiphase kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea to obtain a TMAO-urea titration surface of P protein. Both kinetic and equilibrium studies show evidence of an intermediate state in the P protein folding process. The intermediate state is significantly populated and the folding rate constants involved in the reaction are slow relative to similar size proteins.

NMR spectroscopy was used to characterize the structural properties of the folding intermediate of P protein. The results indicate that the N-terminal (residues 2-19) and C-terminal regions (residues 91-116, 118 is the last residue) are mostly unfolded. 1H-15N HSQC NMR spectra were collected at various pH values. The results suggest that His 22 may play a major role in the energetics of the equilibria between the unfolded, intermediate, and native states of P protein.

Ligand-induced folding kinetics were also investigated to elucidate the overall coupled folding and binding mechanism of P protein and the holoenzyme assembly process. Stopped flow fluorescence experiments were performed at various final ligand concentrations and the data were analyzed using a minimal complexity model that included three conformational states (unfolded, intermediate and folded) in each of three possible liganding states (0, 1 and 2 ligands). The kinetic and equilibrium model parameters that best fit the data were used to calculate the flux through each of the six possible folding/binding pathways. This novel flux-based analysis allows evaluation of the relative importance of pathways in which folding precedes binding or vice versa. The results indicate that the coupled folding and binding mechanism of P protein is strongly dependent on ligand concentration. This conclusion can be generalized to other protein systems for which ligand binding is coupled to conformational changes.

Ligand binding can modulate function of proteins and nucleic acids by changing both the populations of functionally distinct conformational states and the timescales on which they interconvert. For this reason, both thermodynamic and kinetic details of coupling can be important to proper function. How tightly does ligand bind to the different conformational states? What effect does ligand binding have on the conformational equilibrium and conformational kinetics? On what timescales and in what order do binding and conformational change occur? Using a combination of stopped-flow kinetics, isothermal titration calorimetry, and x-ray crystallography, we determine the mechanisms of coupled binding and conformational change in protein (Bacillus subtilis RNase P protein) and RNA (DP17 biosensor) systems.

The results demonstrate that rigorous kinetic analysis can be used to estimate the equilibrium and rate constants for conformational changes, as well as the affinities of ligands for different conformational states. A single ligand can bind to different conformational states of the same protein or nucleic acid with affinities that differ by orders of magnitude. This binding shifts the conformational equilibrium towards the higher affinity state through a combination of increasing rate constants for the forward conformational change and decreasing rate constants for the reverse conformational change. Using a flux-based analysis of the mechanisms we show that molecular recognition is kinetically partitioned between a number of pathways that differ by the order in which binding and conformational change occur. The absolute and relative flux through these pathways varies with ligand concentration, the affinities of the ligand for the various conformational states, and the ability of ligand to accelerate the conformational change. Together, the results give insights into how biological function depends on the kinetic and thermodynamic details of coupled binding and conformational change.

Proteins can form residual compactness in the unfolded state. Among different types of residual compactness, residual helical structure is an important type of local compactness that can propagate through the formation of helical hydrogen bonds. Residual helicity has been observed for different unfolded state proteins. In order to accurately determine the contributions of individual residues to the overall helicity, accurate determination of residue-specific information and quantitative analysis methods are needed.

The projects in this dissertation aim at quantitatively describing the residual helical conformation in the unfolded state of λ-repressor N-terminal domain. The residue-specific helicity values and backbone amide proton hydrogen bonding populations are analyzed using improved methods based on Bayesian inference. Generally, these values are higher for the helix 1 region in the context of the N-terminal domain than as an isolated peptide. Experimentally determined residue-specific helicity values of unfolded state λ-repressor N-terminal domain show similarity to the theoretical prediction using helix-coil model.

These results show that, in the unfolded state of λ-repressor N-terminal domain, the propagation of residual helicity does not significantly depend on tertiary interactions. The results support the hypothesis that λ-repressor N-terminal domain folds by “diffusion-collision”.

Fibronectin (FN) is a large extracellular matrix (ECM) protein that is made up of

type I (FNI), type II (FNII), & type III (FNIII) domains. It assembles into an insoluble

supra-­‐‑molecular structure: the fibrillar FN matrix. FN fibrillogenesis is a cell‐‑mediated process, which is initiated when FN binds to integrins on the cell surface. The FN matrix plays an important role in cell migration, proliferation, signaling & adhesion. Despite decades of research, the FN matrix is one of the least understood supra-­‐‑molecular protein assemblies. There have been several attempts to elucidate the exact mechanism of matrix assembly resulting in significant progress in the field but it is still unclear as to what are FN-­‐‑FN interactions, the nature of these interactions and the domains of FN that

are in contact with each other. FN matrix fibrils are elastic in nature. Two models have been proposed to explain the elasticity of the fibrils. The first model: the ‘domain unfolding’ model postulates that the unraveling of FNIII domains under tension explains fibril elasticity.

The second model relies on the conformational change of FN from compact to extended to explain fibril elasticity. FN contain 15 FNIII domains, each a 7-­‐‑strand beta sandwich. Earlier work from our lab used the technique of labeling a buried Cys to study the ‘domain unfolding’ model. They used mutant FNs containing a buried Cys in a single FNIII domain and found that 6 of the 15 FNIII domains label in matrix fibrils. Domain unfolding due to tension, matrix associated conformational changes or spontaneous folding and unfolding are all possible explanation for labeling of the buried Cys. The present study also uses the technique of labeling a buried Cys to address whether it is spontaneous folding and unfolding that labels FNIII domains in cell culture. We used thiol reactive DTNB to measure the kinetics of labeling of buried Cys in eleven FN III domains over a wide range of urea concentrations (0-­‐‑9M). The kinetics data were globally fit using Mathematica. The results are equivalent to those of H-­‐‑D exchange, and

provide a comprehensive analysis of stability and unfolding/folding kinetics of each

domain. For two of the six domains spontaneous folding and unfolding is possibly the reason for labeling in cell culture. For the rest of the four domains it is probably matrix associated conformational changes or tension induced unfolding.

A long-­‐‑standing debate in the protein-­‐‑folding field is whether unfolding rate

constants or folding rate constants correlate to the stability of a protein. FNIII domains all have the same ß sandwich structure but very different stabilities and amino acid sequences. Our study analyzed the kinetics of unfolding and folding and stabilities of eleven FNIII domains and our results show that folding rate constants for FNIII domains are relatively similar and the unfolding rates vary widely and correlate to stability. FN forms a fibrillar matrix and the FN-­‐‑FN interactions during matrix fibril formation are not known. FNI 1-­‐‑9 or the N-­‐‑ terminal region is indispensible for matrix formation and its major binding partner has been shown to be FNIII 2. Earlier work from our lab, using FRET analysis showed that the interaction of FNI 1-­‐‑9 with a destabilized FNIII 2 (missing the G strand, FNIII 2ΔG) reduces the FRET efficiency. This efficiency is restored in the presence of FUD (bacterial adhesion from S. pyogenes) that has been known to interact with FNI 1-­‐‑9 via a tandem ß zipper. In the present study we

use FRET analysis and a series of deletion mutants of FNIII 2ΔG to study the shortest fragment of FNIII 2ΔG that is required to bind FNI 1-­‐‑9. Our results presented here are qualitative and show that FNIII 2ΔC’EFG is the shortest fragment required to bind FNI 1-­‐‑9. Deletion of one more strand abolishes the interaction with FNI 1-­‐‑9.

Staphylococcus aureus forms biofilms in a variety of human infections. These infections span a wide range of environments in the body. The environment of the infection determines the physiological state of the bacteria. There exists a gap between studying biofilms in a laboratory setting and the physiology of cells in a clinical biofilm. To bridge this gap, we designed a high-throughput biofilm assay to mimic the environment of a clinical infection. Using this assay, we measured the formation of two different classes of biofilms in five clinical isolates. These two classes of biofilms differed in their location: the ring class formed at the air-water interface on the sides of the wells and the bottom class formed at the bottom of the wells. Each class exhibited different phenotypes in response to environmental demands. Biofilms were grown aerobically and anaerobically on plasma-coated surfaces to evaluate the role of environmental oxygen. Protein A (SpA) and polysaccharide amounts were analyzed to elucidate the relationship between proteins, polysaccharides, and biomass accumulation in these two classes of biofilms. The bottom biofilms were disrupted by degradation of polysaccharides, enhanced under hypoxic environments, and their SpA content increased inversely with biomass. In contrast, the ring biofilms were not disrupted by polysaccharide degradation, were enhanced under aerobic environments and their SpA content increased proportionally with biomass. Furthermore, SpA promotion of biofilm formation in ring biofilms was found to be limited to biofilms grown aerobically. Additionally, some results depended on whether the strains were nasal or pathogenic isolates. The results of this study suggest that the influence of environmental and genetic factors on biofilms changes with the physiological state of the biofilm. Because the location of biofilms in S. aureus diseases in patients vary in their environment, our ability to observe these two different classes provides useful insight. This study also evaluated the mechanism by which SpA promotes aerobic ring biofilms. We measured SpA binding to several biofilm matrix components. Isothermal titration calorimetry (ITC) analysis revealed that SpA binds preferentially to β-linked polysaccharides, in particular the major biofilm exopolysaccharide poly-β-1,6-N-acetylglucosamine (PNAG). Biolayer interferometry (BLI) revealed that the multivalency of SpA increases the apparent affinity for β-linked glucans. BLI results also indicated preferential binding of SpA to β-1,6 linked glucans, as shown by ITC, but suggests that SpA can facilitate weaker interactions with the other β-linked glucans. Therefore, we propose that SpA-PNAG binding facilitates cellular aggregation and promotes biofilm formation. Additionally, we proposed that SpA binds other β-linked glucans secreted by other organisms facilitating cellular adhesion in multispecies biofilm infections. Understanding the role of SpA in both single- and multispecies biofilms is important to targeting and combating these types of infections.

An abstract of a thesis devoted to using helix-coil models to study unfolded states.\\

Research on polypeptide unfolded states has received much more attention in the last decade or so than it has in the past. Unfolded states are thought to be implicated in various

misfolding diseases and likely play crucial roles in protein folding equilibria and folding rates. Structural characterization of unfolded states has proven to be

much more difficult than the now well established practice of determining the structures of folded proteins. This is largely because many core assumptions underlying

folded structure determination methods are invalid for unfolded states. This has led to a dearth of knowledge concerning the nature of unfolded state conformational

distributions. While many aspects of unfolded state structure are not well known, there does exist a significant body of work stretching back half a century that

has been focused on structural characterization of marginally stable polypeptide systems. This body of work represents an extensive collection of experimental

data and biophysical models associated with describing helix-coil equilibria in polypeptide systems. Much of the work on unfolded states in the last decade has not been devoted

specifically to the improvement of our understanding of helix-coil equilibria, which arguably is the most well characterized of the various conformational equilibria

that likely contribute to unfolded state conformational distributions. This thesis seeks to provide a deeper investigation of helix-coil equilibria using modern

statistical data analysis and biophysical modeling techniques. The studies contained within seek to provide deeper insights and new perspectives on what we presumably

know very well about protein unfolded states. \\

Chapter 1 gives an overview of recent and historical work on studying protein unfolded states. The study of helix-coil equilibria is placed in the context

of the general field of unfolded state research and the basics of helix-coil models are introduced.\\

Chapter 2 introduces the newest incarnation of a sophisticated helix-coil model. State of the art modern statistical techniques are employed to estimate the energies

of various physical interactions that serve to influence helix-coil equilibria. A new Bayesian model selection approach is utilized to test many long-standing

hypotheses concerning the physical nature of the helix-coil transition. Some assumptions made in previous models are shown to be invalid and the new model

exhibits greatly improved predictive performance relative to its predecessor. \\

Chapter 3 introduces a new statistical model that can be used to interpret amide exchange measurements. As amide exchange can serve as a probe for residue-specific

properties of helix-coil ensembles, the new model provides a novel and robust method to use these types of measurements to characterize helix-coil ensembles experimentally

and test the position-specific predictions of helix-coil models. The statistical model is shown to perform exceedingly better than the most commonly used

method for interpreting amide exchange data. The estimates of the model obtained from amide exchange measurements on an example helical peptide

also show a remarkable consistency with the predictions of the helix-coil model. \\

Chapter 4 involves a study of helix-coil ensembles through the enumeration of helix-coil configurations. Aside from providing new insights into helix-coil ensembles,

this chapter also introduces a new method by which helix-coil models can be extended to calculate new types of observables. Future work on this approach could potentially

allow helix-coil models to move into use domains that were previously inaccessible and reserved for other types of unfolded state models that were introduced in chapter 1.