Browsing by Subject "NMR"

Recent work has shown that hyperpolarized magnetic resonance spectroscopy (HP-MRS) can trace in vivo metabolism of biomolecules and is therefore extremely promising for diagnostic imaging. The most severe challenge this technique faces is the short signal lifetime for hyperpolarization, which is dictated by the spin-lattice (T1) relaxation. In this thesis we show with theory, simulation and experiment that the long-lived nuclear spin states in chemically equivalent or near equivalent spin systems offer a solution to this problem. Spin polarization that has lifetime much longer than T1 (up to 70-fold) has been demonstrated with pulse sequence techniques that are compatible with clinical imaging settings. Multiple classes of molecules have been demonstrated to sustain such long-lived hyperpolarization.

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.

Significance statement

There are two fundamental limitations in magnetic resonance: the poor signal amplitude and the short duration before the system return to equilibrium. Hyperpolarization methods solve the problem of signal amplitude, however, the duration of the hyperpolarized signal is still limited by the spin-lattice relaxation time, T1. Disconnected eigenstates provide a mechanism by which hyperpolarization can be stored for several times T1. This thesis contributes to the knowledge of these states in four important ways. First, the decay of hyperpolarized magnetization of gas is simulated in lung tissue with a contrast agent, yielding insights about the optimal field strength for imaging. Second, I show that it is possible to rapidly discover and characterize disconnected eigenstates by showing that they can be measured without synthesizing the isotopically labeled compound. Third, I extend the spin systems that can support disconnected eigenstates by expanding the theory to include spin-1 nuclei. Finally, I show that disconnected states with long lifetimes can be populated in conjunction with hyperpolarization techniques to simultaneously yield large signal amplitudes for long durations.

Applications of hyperpolarized spin order are likely to be in complex geophysical or biological structures. Understanding the effect of the inhomogeneous fields created when such structures are placed in a magnetic field on hyperpolarized spin order is a necessity to characterize the experimental signal. An example case of hyperpolarized 3He and 129Xe diffusing through lung tissue is examined. In particular a Monte Carlo simulation tool, combined with a magnetic field map of the inhomogeneous field created by mouse lung tissue, is used to determine the dephasing rate of hyperpolarized 3He and 129Xe in the presence of SuperParamagnetic Iron Oxide Nanoparticles (SPION). Contributions to the dephasing rate include the inhomogeneous field, the SPION magnetic field, and dephasing caused by collisions with the confining geometry. The sensitivity of either gas to SPION increases with increasing SPION concentration and decreasing field strength.

There are some general rules about what makes for a disconnected eigenstate (or singlet state) with a long lifetime. However, no systematic experimental study has been undertaken due to the cost and time-constraints of synthesizing the labeled species for study. I show that synthesis is not a barrier for characterizing the long-lived states. Instead the lifetimes may be determined by using the naturally occurring doubly-labeled isotopomer. I verified this method with two compounds, diphenyl acetylene (DPA) and diethyl oxylate (DEO). The former was determined to have a singlet lifetime TS = 251.40 ±3.16 s from the synthesized species, while the naturally occurring isotopomer yielded a lifetime TS = 202 ±55.30 s, both substantially longer than the spin-lattice relaxation time, T1 = 1.63 ±0.01s. In DEO, the lifetime from the disconnected eigenstate was determined to be TS = 14.62 ±0.76 s (synthesized), TS = 19.32 ±3.16 s (naturally occurring). This method is applied to a range of compounds ranging from simple four-spin systems, such as diacetylene (TS = 48.80 ±22.74 s, T1 = 18.66 ±1.16 s) to eight spin systems in dimethylmaleic anhydride (TS = 27.25 ±3.39 s, T1 = 9.38 ±0.43 s). Additionally, a family of compounds including naphthalene (TS = 4.37 ±0.34 s, T1 = 11.33 ±4.89 s), biphenyl (TS = 3.09 ±0.66 s, T1 = 4.69 ±0.10 s), and DPA show that the rotation of the phenyl rings and intermolecular dipole-dipole relaxation can be critical to the relaxation dynamics.

One particular method of accessing the disconnected eigenstate involves coupling a chemically equivalent spin-1/2 pair asymmetrically to an auxiliary spin-1/2 pair. I demonstrate that the disconnected state may still be accessed when the auxiliary nuclei are spin-1. This has two distinct advantages. When the auxiliary nuclei change from proton to deuterium, the couplings are reduced by a factor of ~6.5 which prevents the disconnected state from relaxing as rapidly back to equilibrium. This is demonstrated in diacetylene-d2 and DPA-d10, where the singlet lifetime was extended by a factor of ~1.7 via deuteration (TS,1H = 49 ±23 s, TS,2H = 83 ±30 s for diacetylene and TS,1H = 274 ±6.1 s, TS,2H = 479 ±83 s for DPA). Additionally, by reducing the coupling strength, deuteration allows additional structural moieties to be explored, such as RDC=CDR. One such structure is explored in trans-ethylene-d2, where the singlet character of the protons can be accessed by the reduced coupling to the deuterium. Additionally, this allows for a relatively strong deuterium-deuterium scalar coupling, requiring modification to the theory. This is carried out analytically, and implications for the relaxation properties are performed using a spin-dynamics numerical simulation. The lifetime of the disconnected state was determined to be TS = 30.2 ±12.3 s, compared to the T1 = 1.1 ±0.2 s at high concentration (270 mM), and increasing to TS = 117. ±9.80 s at low concentration (52 mM). The variation in long lifetime is attributed to intermolecular dipole-dipole relaxation.

Ultimately, the gains in lifetime from using disconnected eigenstates provide a means to the practical implementation of hyperpolarization in a wider range of experiments. A recent hyperpolarization method, Signal Amplification By Reversible Exchange in Shield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) is shown to directly hyperpolarize long lived spin order in a diazirine containing molecule. Diazirine rings are three member N=N-C groups that can replace a methylene group and serve as a versatile MR and optical molecular tag. Hyperpolarization is accomplished by bubbling parahydrogen through a solution containing the diazirine and an iridium catalyst. Due to the chemical inequivalence of the 15N of the diazirine, hyperpolarization of longitudinal magnetization and singlet character could be observed by transfer to the high field spectrometer. Signal enhancements of over 14,000 were observed. The magnetic field strength required for buildup of magnetization and singlet character was derived and is in agreement with the experiment. The magnetization lifetime was observed to be T1 = 5.75 ±0.18 minutes and independent of field strength, while the lifetime of the singlet character was observed to be as long as TS = 30.1 ±13.4 minutes at low field (3 Gauss).

The combination of these experiments – understanding lifetimes in inhomogeneous magnetic fields that will be encountered in experiment, identification of disconnected eigenstates with long lifetimes via the naturally occurring isotopomer and extending these lifetimes even further with deuteration, and finally, the direct generation of long-lived hyperpolarized spin order – allows a measurement that required hyperpolarized spin order for the enhanced signal amplitude, to be carried out.

Although microbes may strongly influence wetland phosphorus (P) cycling, specific microbial communities and P metabolic processes have not been characterized in wetlands, and microbial P cycling is poorly understood across global ecosystems, especially in soils. The goal of this work is to test the effects of stress and growth factors on microbial communities in wetlands, and on microbial P metabolism and P cycling at ecosystem scales in wetland soils and beyond. I conducted field and laboratory research experiments in wetland soils, which by definition lie along gradients between terrestrial and aquatic ecosystems, and I explicitly compared results in wetlands to adjacent ecosystems to improve inference and impact.

To test relationships between microbial communities, soil stress and resource supply, I compared the distribution and abundance of uncultured bacterial communities to environmental factors across a range of wetland soils including a well-characterized P enrichment gradient, and restoration sequences on organic soils across freshwater wetland types. The strongest predictor of bacterial community composition and diversity was soil pH, which also corresponded with the abundance of some bacterial taxa. Land use and restoration were also strong predictors of bacterial communities, diversity, and the relative abundance of some taxonomic groups. Results from wetland soils in this study were similar to both terrestrial and aquatic ecosystems in the relationship of pH to microbial communities. However, patterns of biogeography I observed in wetlands differed from aquatic systems in their poor relationships to nutrient availability, and from terrestrial ecosystems in the response of microbial diversity to ecosystem restoration.

Accumulation of inorganic polyphosphate (PolyP) is a critical factor in the survival of multiple environmental stresses by bacteria and fungi. This physiological mechanism is best characterized in pure cultures, wastewater, sediments, and I used 31P-NMR experiments to test whether similar processes influence microbial P cycling in wetland soils. I surveyed PolyP accumulation in soils from different wetland types, and observed PolyP dynamics with flooding and seasonal change in field soils and laboratory microcosms. I found PolyP accumulation only in isolated pocosin peatlands, similar to patterns in the published literature. I observed rapid degradation of PolyP with flooding and anerobic conditions in soils and microcosms, and I characterized the biological and intracellular origin of PolyP with soil cell lysis treatments and bacterial cultures. While degradation of PolyP with flooding and anaerobic conditions appeared consistent with processes in aquatic sediments, some seasonal patterns were inconsistent, and experimental shifts in aerobic and anaerobic conditions did not result in PolyP accumulation in soil slurry microcosms. Similar to patterns in wetlands, I found prior observations of PolyP accumulation in published 31P-NMR studies of terrestrial habitats were limited to acid organic soils, where PolyP accumulation is thought to be fungal in origin. Fungal accumulation of PolyP may be useful as an alternative model for PolyP accumulation in wetlands, although I did not test for fungal activity or PolyP metabolism.

To evaluate relationships between microbial P metabolism and growth, I compared concentrations of P in soil microbial biomass with the soil metabolic quotient (qCO2) by compiling a large-scale dataset of the carbon (C), nitrogen (N) and P contents of soils and microbial biomass, along with C mineralization rates across global wetland and terrestrial ecosystems (358 observations). The ratios of these elements (stoichiometry) in biomass may reflect nutrient limitation (ecological stoichiometry), or be related to growth rates (Biological Stoichiometry). My results suggest that the growth of microbial biomass pools may be limited by N availability, while microbial metabolism was highly correlated to P availability, which suggests P limitation of microbial metabolism. This pattern may reflect cellular processes described by Biological Stoichiometry, although microbial stoichiometry was only indirectly related to respiration or metabolic rates. I found differences in the N:P ratios of soil microbial biomass among ecosystems and habitats, although high variation within habitats may be related to available inorganic P, season, metabolic states, or P and C rich energy storage compounds. Variation in microbial respiration and metabolic rates with soil pH suggests important influences of microbial communities and their responses to stress on metabolism and P cycling.

My dissertation research represents early contributions to the understanding of microbial communities and specific processes of microbial P metabolism in wetlands, including PolyP accumulation and Biological Stoichiometry, which underpin microbial cycling of P and C. Together, my research findings broadly indicate differences in microbial P metabolism among habitats in wetlands and other ecosystems, which suggests the prevailing paradigm of uniform P cycling by microbes will be inadequate to characterize the role of microbes in wetland P cycling and retention. While I observed some concomitant shifts in microbial communities, PolyP accumulation, and microbial stoichiometry with soil pH, land use, and habitat factors, relationships between specific microbial groups and their P metabolism is beyond the scope of this work, but represents an exciting frontier for future research studies.

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”.

A molecular depiction of the assembly, interaction and regulation of protein complexes is essential to the understanding of biological functions of protein complexes. Structural analysis of protein complexes by Nuclear Magnetic Resonance (NMR) has relied heavily on the detection and assignment of intermolecular Nuclear Overhauser Effects (NOEs) that define the interactions of protons at the molecular interface. Intermolecular NOEs have traditionally been detected from 3-D half-filtered NOE experiments by suppressing intramolecular NOEs prior to NOE transfer. However, due to insufficient suppression of undesirable signals and a lack of dispersion in the H dimension, data analysis is complicated by the interference of residual intramolecular NOEs and assignment ambiguity, both of which can lead to distorted or even erroneously packed protein complex structures. Leveraging the recent development of fast NMR technology based on sparse sampling in our lab, we developed a strategy for reliable identification and assignment of intermolecular NOEs using high resolution 4-D NOE difference spectroscopy. Spectral subtraction of individually labeled components from a uniformly labeled protein complex yields an "omit" spectrum containing only intermolecular NOEs with little signal degeneracy.

The benefit of such a strategy is first demonstrated in structural analysis of a homooligomeric protein complexes, the foldon trimer. We show that intermolecular NOEs collected from the 4-D omit NOE spectrum can be directly utilized for automated structural analysis of the foldon trimer by CYANA, whereas intermolecular NOEs derived from 3-D half-filtered NOE experiments failed to generate a converged structure under the same condition.

Such a strategy was further demonstrated on a heterodimeric protein complex in translesion sysnthesis (TLS), a DNA damage tolerance pathway. The TLS machinery consists of several translesion DNA polymerases that are recruited to the stalled replication fork in response to monoubiquitinated proliferating cell nuclear antigen (PCNA) in order to bypass DNA lesions encountered during genomic replication. The recruitment and assembly of translesion machinery is heavily dependent on ubiquitin-binding domains, including ubiquitin-binding motifs (UBMs) and ubiquitin-binding zinc fingers (UBZs) that are found in translesion DNA polymerases. Two conserved ubiquitin-binding motifs (UBM1 and UBM2) are found in the Y-family polymerase (Pol) &iota, both of which contribute to ubiquitin-mediated accumulation of Pol &iota during TLS. Although the Pol&iota UBM2-ubiquitin complex has been previous reported by our lab and others, the Pol &iota UBM1-ubiquitin complex has remained a challenge due to significant signal overlap in conventional 3-D NOE spectroscopy. In order to determine the molecular basis for ubiquitin recognition of Pol &iota, we solved the structures of human Pol &iota UBM1 and its complex with ubiquitin by 4-D fast NMR, revealing a signature helix-turn-helix motif that recognizes ubiquitin through an unconventional surface centered at L8 of ubiquitin. Importantly, the use of 4-D omit NOE spectroscopy unambiguously revealed an augmented ubiquitin binding interface that encompasses the C-terminal tail of UBM1.

4-D omit NOE spectroscopy was also used to study the Fanconi anemia associated protein 20 (FAAP20)-ubiquitin complex within the Fanconi Anemia (FA) complexes required for efficient repair of DNA interstrand crosslinks (ICLs), a process that is mediated by the ubiquitin-binding zinc finger (UBZ) domain of FAAP20. Unexpectedly, we show that the FAAP20-ubiquitin interaction extends beyond the compact UBZ module and is accompanied by transforming the disordered C-terminal tail of FAAP20 into a rigid &beta-loop, with the invariant C-terminal tryptophan (W180 of human FAAP20) emanating toward I44 of ubiquitin for enhanced binding. Accordingly, alanine substitution of the absolutely conserved C-terminal tryptophan residue of FAAP20 abolishes ubiquitin binding and impairs FA core complex-mediated ICL repair in vivo.

Reliable detection and unambiguous assignment of intermolecular NOEs is essential to NMR-based structure determination of protein complexes. The development of 4-D omit NOE spectroscopy in this thesis overcomes many limitations of conventional 3-D half-filtered experiments to allow for reliable detection and unambiguous assignment of intermolecular NOEs of heterodimeric complexes and homooligomeric complexes. These advantages render such a strategy particularly attractive for structural studies of protein complexes by biomolecular NMR.

Bulges are ubiquitous building blocks of the three-dimensional structure of RNA. They help define the global structure of helices and points of flexibility allowing for functionally important dynamics, such as binding of proteins, ligands and small molecules to occur. This thesis utilizes a battery of nuclear magnetic resonance (NMR) methods and a model system of RNA bulge motifs, the transactivation response element (TAR) RNA from the human immunodeficiency virus type 1 (HIV-1), to characterize the dynamic energy landscape of bulges. Specifically investigating how it varies with bulge length, divalent cations, and in the presence of epi-transcriptomic modifications.

Deleting a single bulge residue (C24) from trinucleotide HIV-1 TAR bulge shifts a pre-existing equilibrium from the unstacked to a stacked conformation in which the bulge residues flip out of the helix and are highly flexible at the picosecond-to-nanosecond timescale. However, the mutation minimally impacts microsecond-to-millisecond conformational exchange directed towards two low-populated and short-lived excited conformational states that form through a reshuffling of bases pairs throughout TAR. The mutant does, however, adopt a slightly different excited conformational state on the millisecond timescale. Therefore, minor changes in bulge topology preserve motional modes occurring over the picosecond-to-millisecond timescales but alter the relative populations of the sampled states or cause subtle changes in their conformational features.

The impact of more broadly varying the length of the TAR poly-pyrimidine bulge (n = 1, 2, 3, 4 and 7) on inter-helical dynamics has been studied across a range of Mg2+ concentrations. In the absence of Mg2+ (25 mM monovalent salt), n 3 bulges adopt predominantly unstacked conformations (stacked population <15%) whereas 1-bulge and 2-bulge motifs adopt predominantly stacked conformations (stacked population >85%). The 2-bulge motif is biased toward linear conformations and increasing the bulge length leads to broader inter-helical distributions and structures that are on average more kinked. In the presence of 3 mM Mg2+, the helices predominantly coaxially stack (stacked population >75%), regardless of bulge length, and the midpoint for the Mg2+-dependent stacking transition does not vary substantially (within 3-fold) with bulge length. In the absence of Mg2+, the difference between the free energy of inter-helical coaxial stacking across the bulge variants is estimated to be ~2.9 kcal/mol, based on an NMR chemical shift mapping approach, with stacking being more energetically disfavored for the longer bulges. This difference decreases to ~0.4 kcal/mol in the presence of 3 mM Mg2+. It is proposed that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state with increasing bulge length thus increasing the number of co-axial conformations that can be sampled.

N6-Methyladenosine (m6A) and N1-Methylpurine (m1A and m1G) xx or just refer to m1G?xx are post-transcriptional RNA modifications that are proposed to influence RNA function through mechanisms that can involve modulation of RNA structure. m6A is thought to modulate RNA structure by destabilizing base pairing. Here, it is shown that m6A can stabilize A-U base pairing and overall RNA structure when placed within the context of a bulge motif. m1A has also been shown to potently destabilize RNA duplexes due to their inability to favorably accommodate Hoogsteen base pairing. It is shown that such Hoogsteen base pairs can form in RNA when placed in the context of a bulge motif.

Taken together, the studies show that the dynamic energy landscape of polypyridine bulges is highly robust with respect to changes in bulge length allowing for gradual variations in the population and energetics of common conformations. Mg2+ plays an important role in smoothening these variations most likely by diminishing electrostatic contributions that could vary significantly across bulges of different length. The results also show that the structural impact of epi-transcriptomic modifications can be greatly altered relative to duplex RNA when targeting bulge motifs.

Ssu72 is a protein phosphatase that selectively targets phosphorylated serine residues at the 5th position (pS5) in the heptad repeats of the C-terminal domain (CTD) of RNA polymerase II, in order to regulate the CTD-mediated coupling between eukaryotic transcription and co-transcriptional events. The biological importance of Ssu72 is underscored by (1) the requirement of its activity for viability in yeast, and (2) the numerous phenotypes - affecting all three stages of the transcription cycle - that result from its mutation in yeast. Despite limited homology to the low molecular weight (LMW) subclass of protein tyrosine phosphatases (PTPs), several lines of evidence suggest that Ssu72 represents the founding member of a new class of enzymes, including its unique substrate specificity and an in vivo connection with the activity of proline isomerase Ess1.

The main focus of this thesis has been to structurally and kinetically characterize Ssu72, in order to define its relation to known enzyme families, to provide biochemical explanations for extant in vivo observations, and to allow future structure-guided investigations of its role in coordinating transcription with co-transcriptional events. To this end, we solved the structure of Ssu72 in complex with its pS5 CTD substrate, revealing an enzyme fold with unique structural features and a surprising substrate conformation with the pS5-P6 motif of the CTD adopting the cis configuration. Together with kinetic assays, the structure provides a new interpretation of the role of proline isomers in regulating the CTD phosphorylation state, with broad implications for CTD biology.

The second goal of this thesis has been to develop new methods for NMR studies of large proteins, which present unique challenges to conventional methods, including fast signal decay and severe signal degeneracy. The first of these new methods, the `just-in-time' HN(CA)CO, improves the sensitivity of a common backbone assignment experiment. The next two methods, the 4-D diagonal-suppressed TROSY-NOESY-TROSY and the 4-D time-shared NOESY, were designed for use with sparse sampling techniques that allow the acquisition of high-resolution, high-dimensionality datasets. These efforts culminate with global fold calculations for large proteins, including the 23 kDa Ssu72, with accurate and unambiguous automated assignment of NOE crosspeaks. We expect that the methods presented here will be particularly useful as the NMR community continues to push toward higher molecular weight targets.

As non-coding RNAs are increasingly implicated in cellular regulatory functions and disease states, there is a need to deepen our understanding of RNA structure-function relationships as well as to develop methods targeting RNA with small molecules. The transactivation response element (TAR) RNA from human immunodeficiency virus type 1 (HIV-1) is an established drug target for the development of anti-HIV therapeutics and has served as a model system for understanding RNA dynamics and RNA:ligand interactions. Like many RNAs, HIV-1 TAR is a highly flexible molecule that experiences dynamics ranging from local fluctuations in base orientation and interhelical angles to higher-order dynamics that transiently alter base pairing away from the ground state (GS) secondary structure. The work presented in this thesis is aimed at developing approaches targeting TAR with small molecules that integrate its broad range of structural dynamics.

First, nuclear magnetic resonance (NMR) chemical shift mapping is applied in concert with fluorescence binding assays and computational docking to efficiently characterize the TAR-binding modes of a focused library of amiloride derivatives. Through this work, amiloride is established as a novel RNA binding scaffold with interesting structure-activity relationships. Ultimately, this approach yielded ten novel TAR binders with demonstrated selectivity for TAR over tRNA and with up to a 100-fold increase in activity over the parent dimethyl amiloride compound.

Next, we demonstrate that ensemble-based virtual screening (EBVS) is a powerful approach to predict ligand binding for flexible RNA targets. Here, we generate a library to evaluate EBVS enrichment by subjecting HIV-1 TAR to experimental high-throughput screening against ~100,000 drug-like small molecules. EBVS against a dynamic ensemble of the TAR GS determined previously by combining NMR spectroscopy data and molecular dynamics (MD) simulations scores hits and non-hits with an area under the receiver operator characteristic curve of ~0.85-0.94 and with ~40-75% of all hits falling within the top 2% of scored molecules. Importantly, the enrichment was shown to depend on the accuracy of the ensemble.

Finally, we explore the novel strategy of specifically targeting non-native RNA excited state conformations inspired by the fact that their altered secondary structures are likely functionally inactive and highly unique. We use a mutational stabilize-and-rescue approach to demonstrate that TAR ES2 dramatically inhibits TAR activity in cells, suggesting that stabilizing the ES conformation with small molecules would similarly inhibit activity. To pursue TAR ES2 as a potential target, we have determined the first-ever dynamic ensemble of an RNA ES using a combination of MD and NMR residual dipolar couplings (RDCs) measured on a highly accurate ES2-stabilizing mutant. This dynamic ensemble was subjected to our validated EBVS approach to identify small molecules that bind and stabilize TAR ES2. Using NMR chemical shift fingerprinting, we have identified molecules that bind the TAR ES2 structure, including two that induce significant broadening in wtTAR consistent with chemical exchange and two that show a preference for TAR ES2 over the GS.

Together, this work explores multiple novel strategies for structure-specific RNA targeting.

We present a computational structure-based redesign of the phenylalanine adenylation domain of the non-ribosomal peptide synthetase (NRPS) enzyme gramicidin S synthetase A (GrsA-PheA) for a set of non-cognate substrates for which the wild-type enzyme has little or virtually no specificity. Experimental validation of a set of top-ranked computationally-predicted enzyme mutants shows significant improvement in the specificity for the target substrates. We further present enhancements to the methodology for computational enzyme redesign that are experimentally shown to result in significant additional improvements in the target substrate specificity. The mutant with the highest activity for a non-cognate substrate exhibits 1/6 of the wild-type enzyme/wild-type substrate activity, further confirming the feasibility of our computational approach. Our results suggest that structure-based protein design can identify active mutants different from those selected by evolution.

Knowledge about the structures of individual domains and domain interactions can further our redesign of the NRPS enzymes for new bioactive nature product. So far, little structure information has been available for the auxiliary domains such as the epimerization domains and how they interact with the NRPS modules. Solution structure studies by nuclear magnetic resonance (NMR) provide advantages for understanding the dynamics of the domains and reveal active conformations that sometimes are not represented by the crystal structures. However, the large size of the NRPS proteins present challenges for structure studies in solution. In chapter 3, we study the solution structure of the 56 kDa epimerization domain of GrsA (GrsA-PheE) by NMR. We use multidimensional backbone resonance experiments as well as specific labeling strategy to assign the backbone resonances of GrsA-PheE. Secondary structures are determined by sets of residual dipolar couplings (RDCs) measured in multiple alignment media. To determine the global fold of the protein, we obtain long-range distance restraints by measuring the paramagnetic relaxation enhancements (PREs) from 15 site-directed spin labeling samples.

In chapter 4, we investigate the redox regulation of phosphatases. The activity levels of protein tyrosine phosphatases (PTPs) in cells are highly regulated in various ways including by phosphorylation, localization and protein-protein interaction. Additionally, redox-dependent modification has emerged as a critical part in attenuating PTPs activity in response to cellular stimuli. The tandem Src homology 2 domain-containing PTPs (SHPs) belong to the family of nonreceptor PTPs. The activity level of SHPs is highly regulated by interaction of SH2 domain, phosphorylation level of C-terminal tail and by reversible oxidation. In vivo evidence has shown the reversible oxidation of catalytic cysteine inhibits SHPs activity transiently as a result, affecting the phosphorylation level of its target proteins. In this chapter, we investigate in vitro the reversible oxidation of full-length and catalytic domain of SHP-1 and SHP-2 by using kinetic measurements and mass spectrometry. We have confirmed the susceptibility of the active site cysteines of SHPs to oxidative inactivation, with rate constants for oxidation similar to other PTPs (2-10 M-1s-1). Both SHP-1 and SHP-2 can be reduced and reactivated with the reductants DTT and gluthathione, whereas only the catalytic domain of SHP-2 is subject to reactivation by thioredoxin. Unlike PTPs whose oxidation contains a catalytic cysteine disulfide bonding to a backdoor cysteine or forms a sulfenylamide bonding to nearby backbone nitrogen, we have found that in the reversibly oxidized SHPs, the catalytic cysteines is re-reduced while two conserved backdoor cysteines form a disulfide linkage. Knocking out either of the backdoor cysteine preserves the reversibility of the oxidized SHPs with a disulfide formation between the catalytic cysteine and the remaining backdoor cysteine. However, removal of both backdoor cysteines leads to irreversible oxidative inactivation, demonstrating that these two cysteines are necessary and sufficient for ensuring reversible oxidation of the SHPs. Our results extend the mechanisms by which redox regulation of PTPs is used to modulate intracellular signaling pathways.

The central dogma of molecular biology relies on the correct Watson-Crick (WC) geometry of canonical deoxyribonucleic acid (DNA) dG•dC and dA•dT base pairs to replicate and transcribe genetic information with speed and an astonishing level of fidelity. In addition, the Watson-Crick geometry of canonical ribonucleic acid (RNA) rG•rC and rA•rU base pairs is highly conserved to ensure that proteins are translated with high fidelity. However, numerous other potential nucleobase tautomeric and ionic configurations are possible that can give rise to entirely new pairing modes between the nucleotide bases. Very early on, James Watson and Francis Crick recognized their importance and in 1953 postulated that if bases adopted one of their less energetically disfavored tautomeric forms (and later ionic forms) during replication it could lead to the formation of a mismatch with a Watson-Crick-like geometry and could give rise to “natural mutations.”

Since this time numerous studies have provided evidence in support of this hypothesis and have expanded upon it; computational studies have addressed the energetic feasibilities of different nucleobases’ tautomeric and ionic forms in siico; crystallographic studies have trapped different mismatches with WC-like geometries in polymerase or ribosome active sites. However, no direct evidence has been given for (i) the direct existence of these WC-like mismatches in canonical DNA duplex, RNA duplexes, or non-coding RNAs; (ii) which, if any, tautomeric or ionic form stabilizes the WC-like geometry. This thesis utilizes nuclear magnetic resonance (NMR) spectroscopy and rotating frame relaxation dispersion (R1ρ RD) in combination with density functional theory (DFT), biochemical assays, and targeted chemical perturbations to show that (i) dG•dT mismatches in DNA duplexes, as well as rG•rU mismatches RNA duplexes and non-coding RNAs, transiently adopt a WC-like geometry that is stabilized by (ii) an interconnected network of rapidly interconverting rare tautomers and anionic bases. These results support Watson and Crick’s tautomer hypothesis, but additionally support subsequent hypotheses invoking anionic mismatches and ultimately tie them together. This dissertation shows that a common mismatch can adopt a Watson-Crick-like geometry globally, in both DNA and RNA, and whose geometry is stabilized by a kinetically linked network of rare tautomeric and anionic bases. The studies herein also provide compelling evidence for their involvement in spontaneous replication and translation errors.