As an RNA-guided RNA modification enzyme, the box H/ACA RNP recruits the guide RNA to recognize the substrate RNA, whereas the protein partners carry out the catalysis. Most intriguingly, box H/ACA RNPs share the same four conserved proteins, Cbf5, Nop10, L7Ae and Gar1, and are able to utilize more than 100 guide RNAs to guide posttranscriptional modifications in most stable RNAs. With respect to structural studies, archaeal box H/ACA ribonucleoproteins (RNPs) are probably one of the most extensively characterized ribonucleoprotein particles to this day. The dissertation presented here describes this work's contribution to the understanding of the nature of the RNA-guided RNA modification enzyme and of the most complex pseudouridine synthases. In chapter 2, we explored the structural basis of an archaeal box H/ACA protein complex comprised of three of the four essential proteins, Cbf5, Nop10 and Gar1. It was the first time we obtained molecular insights into these three proteins and the implications of a severe disease called dyskeratosis congenita (DC). We have also identified a DC mutation cluster site within a modeled dyskerin (Cbf5 homolog in humans) structure. In chapter 3, we further characterized the three-dimensional structure of a catalytically deficient archaeal box H/ACA RNP complex, including the guide RNA, the substrate RNA, Cbf5, Nop10 and Gar1. We devised a non-intrusive 2-aminopurine (2-AP) fluorescence assay which allowed us to determine the precise placement of the target uridine at the active site requires a conformational change of the guide-substrate RNA duplex by L7Ae. In chapter 4, we further examined the structural basis for accurate placement of substrate by accessory proteins using the 2-AP fluorescence assay. Our results revealed that each of the three accessory proteins, Nop10, L7Ae and Gar1, as well as an active site residue, have distinct effects on substrate conformations, suggesting the cooperative network of box H/ACA RNP. In chapter 5, we described a substrate-bound functional archaeal box H/ACA RNP that revealed detailed information about the active site. The substrate RNA containing 5-fluoruridine at the modification position is fully docked and rearranged in a manner similar to those of stand-alone pseudouridine synthases. The complementary biochemical studies further revealed the importance of a conserved protein loop and a guide-substrate RNA pocket in the binding to the substrate. With further comparison of available structures of stand-alone pseudouridine synthases-RNA complexes, we are able to summarize the common mechanism among all pseudouridine synthases, perhaps also a theme in other widespread RNA-guided enzymes. The accomplishments of this work greatly enhance our understanding of the enzymatic architecture of box H/ACA RNPs, unravel the many intriguing features of the most complex pseudouridine synthases, and shed light on the nature of the RNA-guided RNA modification and the assembly architecture of the telomerase holoenzyme. / A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2009. / July 23, 2009. / Includes bibliographical references. / Hong Li, Professor Directing Dissertation; Igor Alabugin, Outside Committee Member; Kenneth A. Taylor, Committee Member; Hank W. Bass, Committee Member; Richard Bertram, Committee Member.

Muscle contraction is regulated at the thin filament level by tropomyosin (Tm) and the troponin complex (Tn). Tm is an α-helical coiled coil protein that forms a continuous strand on the actin filament through end-end interactions with neighboring Tm. Tn is a heterotrimer consisting of a calcium (Ca2+) binding subunit (TnC), a thin filament binding inhibitory subunit (TnI) and a Tm binding subunit (TnT). A postulated model for activation of muscle contraction states that upon Ca2+-binding to TnC, TnC opens up a hydrophobic patch to which the switch peptide of TnI binds. The movement of the TnI switch peptide towards the hydrophobic patch of TnC pulls the TnI inhibitory peptide with it. The movement of the TnI inhibitory peptide relieves the myosin-binding site on actin allowing myosin heads to bind weakly. This leads to the positioning of Tm into the actin groove further exposing the myosin binding sites on actin and allowing myosin heads to bind strongly to actin and contraction to take place. Many aspects of this model of the regulation are still debatable. In the first aim (Chapter 3), we are studying what changes Tm position on the surface of the actin filament to hide or expose the myosin-binding sites on actin. One previous hypothesis was that Tm dynamics (i.e. variations in the amplitude of Tm vibrations) might drive the change in its radial position on actin. We show, using saturation transfer electron paramagnetic resonance (ST-EPR) that Tm does not change its dynamics dramatically between the blocked, closed and open states and thus Tm's dynamics is not a main player in the change of Tm's position on the actin filament. The study was done on four different positions on Tm (N-terminus, middle, Tn binding region and C-terminus). The rates of motion varied along the length of tropomyosin with the C-terminus position being one order of magnitude slower than the N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber. In the second aim (Chapter 4), we study how the C-terminal domain of TnI regulates acto-myosin contraction. It has been shown that several mutations in the C-terminal domain of TnI lead to various cardiomyopathies, which is an indication that the C-terminal domain of TnI is essential for regulation of muscle contraction. Indeed, in addition to the well-established role of TnI's inhibitory peptide (skeletal residues 104-115, cardiac residues 137-148) in the regulation of muscle contraction, the C-terminal domain of TnI (skeletal residues 116-182, cardiac residues 149-210) has been shown to be important for full inhibition of muscle contraction. In this study, we determined the backbone dynamics of the C-terminal domain of TnI using EPR. In the troponin complex that is free to tumble in solution the correlation time was 1.5 ns as expected for a ~6 kDa tethered domain. However, when reconstituted into "ghost" muscle fiber (containing actin and Tm) the motion was slowed to microseconds indicating strong interaction with the thin filament. The dynamics of the C-terminal domain of TnI was five-fold slower in the absence of Ca2+ (140 μs) than in its presence (30 μs) indicating increased interaction with actin/Tm in the off state. In addition, Förster Energy Transfer (FRET) was used to position the C-terminal domain of TnI with respect to actin and Tm in the reconstituted thin filament. Our data indicate a small change in the position of the C-terminal domain of TnI (TnI 190, 197 and 210) away from actin 374 and Tm 146 upon Ca2+ binding. The position of one of the mutants of TnI (TnI 197) was placed within the constraints of a recently proposed actin-Tm model. Two areas that are symmetrical with respect to the Tm long axis were found to satisfy our restraints. Combining the position and dynamics results we conclude that the C-terminal domain of TnI does not significantly change its position from one state to another but significantly alters its dynamics. / A Dissertation submitted to the Molecular Biophysics Program in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2011. / September 12, 2011. / bi-functional spin label, electron paramagnetic resonance, Forster resonance energy transfer, muscle, Tropomyosin, Troponin / Includes bibliographical references. / Piotr G. Fajer, Professor Directing Dissertation; Peng Xiong, University Representative; Timothy M. Logan, Committee Member; P. Bryant Chase, Committee Member; Geoffrey F. Strouse, Committee Member.

In eukaryotes and archaea 5-25% of transfer RNA (tRNA) precursors contain intervening sequences, or introns, that interrupt the molecules' functional regions. Because functional tRNA molecules are necessary for protein synthesis, removing these introns is essential to sustain life. tRNA introns are removed in a two-to-three step process mediated by three different proteins. The RNA splicing endonuclease acts first to cleave two phosphodiester bonds at the intron boundaries within the folded precursor RNAs. The endonuclease performs this function upon nuclear tRNA introns and all archaeal introns. It is well-established that in all organisms the endonuclease step in the splicing pathway is completely conserved, with evidence for the conservation of cleavage chemistry being provided by biochemical studies. However, no detailed information was previously available regarding the endonuclease's specific mechanisms. This research addresses two key aspects of the splicing endonuclease mechanism, namely, substrate recognition and catalysis. Chapter 2 explores the structural elements in a phenotypical archaeal splicing endonuclease and its RNA substrate required for recognition and catalysis. These assays explicitly demonstrate the enzyme and substrate elements involved in recognition and binding. They also support previous findings regarding a conserved triad hypothesized to be catalytic and lay the foundation for the more in-depth studies in Chapter 3. Chapter 3 presents a series of kinetics experiments investigating this conserved triad in which kinetic parameters KM and k2 are obtained. The primary substrate recognition elements in the endonuclease are strictly conserved. However, splicing endonucleases in different organisms are found to have different subunit compositions and substrate specificities. No biochemical studies to date have shed light on how this occurs. Chapter 4 presents studies exploring how the enzyme's quaternary structure affects substrate recognition and cleavage. These studies are continued in Chapter 5, where it is demonstrated that enzyme assembly alone can dictate both substrate specificity and activity. Taken in total, the work presented in this Dissertation provides significant insight regarding how the endonuclease precisely recognizes intron-exon junctions and accelerates the cleavage reaction. It also sheds considerable light on how enzyme subunit composition and quaternary structure relate to the mechanism of RNA recognition. / A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2007. / April 3, 2007. / Splicing Endonuclease, RNA-Protein Interactions, RNA, Structure-Function Studies, tRNA / Includes bibliographical references. / Hong Li, Professor Directing Dissertation; Myra Hurt, Outside Committee Member; Timothy Cross, Committee Member; Timothy Logan, Committee Member; Brian Miller, Committee Member.

Muscle plays a primary role in movement of the body of multicellular organs. A study of muscle contraction at a molecular level will provide understanding of muscular malfunction, as well as insights into the basic mechanism of bimolecular motors. Muscle contraction involves a complex interaction between multiple proteins with multiple domains. Not all of these interactions are well understood. This study is focused on the role of the S2 segment of muscle myosin. S2 is part of the long alpha-helix coiled-coil rod, and plays a significant role in both muscle contraction and myosin II ATPase regulation. In this study, we use the HMM fragment of smooth muscle myosin II (smHMM) which contains a pair of myosin heads held together by the S2 domain. smHMM with a full length S2 shows the ATPase behavior of a fully regulated smooth muscle myosin (smM) but is soluble rather than filamentous. Biochemical studies have shown that the length of the S2 domain affects actin-activated ATPase regulation in smHMM, an observation that would suggest that torsional rigidity of the alpha-helices that comprise S2 is the physical basis because the two heads, which would be nominally on opposite sides of the coiled-coil alpha-helices, must rotate to a position on one side only. However, interpretation of the biochemistry suggested that the effect of S2 length on regulation was due to a requirement that the S2 coiled-coil be long enough to interact with one of the myosin heads in the inactive complex. Modeling studies investigating this effect concluded that torsional rigidity was the explanation. Torsional motions could also be involved in the binding of both myosin heads to a single actin filament because as with formation of the ATPase inhibited conformation, the two heads must come to the same side of the S2 domain. In this case, there is no suggestion of an interaction between the S2 domain and the actin-attached heads and thus an effect can have a simpler interpretation. On that basis, we investigated whether the length of the S2 domain has an effect on the simultaneous binding of both myosin heads to an actin filament using two recombinant smHMM constructs, one with a full length S2 (wt-HMM), the other with a highly shortened S2 with a length of two heptads of S2 followed by a 32 residue leucine zipper (2hepzip-HMM). We compare the amount of 2-headed binding to actin in these two otherwise identical constructs in the complete absence of nucleotide, known as the rigor state, and in the presence of saturating levels of ADP. The myosin S1 head has its strongest binding affinity to actin in the rigor state; while in the presence of ADP, it has less binding affinity. Thus, if torsional rigidity is the physical basis of the effect, the predicted outcome is more 2-headed attachment to actin with wt-HMM-rigor compared to 2hepzip-HMM-rigor, and a less 2-headed attachment for both constructs when ADP is added. For this study, we directly visualize the smHMM attachment to actin filament by combining cryo-electron tomography with subvolume alignment and classification using multivariate data analysis. Methodological advances in several steps were necessary to achieve this goal: (1) segmentation of the subvolumes from the tomograms, (2) alignment of the subvolumes to a feature held in common among all members, and (3) clustering into groups a heterogeneous collection of subvolumes that vary with respect to several criteria. In this study the common feature is the actin filaments, and the heterogeneous feature is the presence or absence of myosin binding to actin by either one head or two. We use a novel approach that utilizes convolution and least squares fitting to smooth the stochastic error in the subvolume centers and Euler angles to improve the alignment. The subvolume alignment was done in a way that enabled all the bound myosin heads to be localized to a single classification site from which the variability can be assessed in a simplified way. Following identification of the bound myosin heads, the different types of attachment are determined that include 2-headed attachments, 1- headed attachments with one free head, which may or may not have an accessible, unlabeled actin subunit nearby, and 1-headed attachments that have no unbound second head, which may arise from dissociation of the S2 domain (Chapter 3). We find that the when the S2 domain is shortened to a length of 2 heptads plus a leucine zipper that 2-headed binding decreases by a factor of 2 compared to full length S2 constructs. This we interpret as evidence of a torsional effect of the S2 helices. Moreover, the addition of ADP, rather than decreasing the amount of 2-headed binding increased it by ~5% in the case of wt-HMM. The effect of ADP addition is compatible with published accounts of ADP addition to smooth muscle fibers, which showed that tension increased by ~3% and stiffness decreased by ~10% (Chapter 4). / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2014. / November 12, 2014. / cryo-electron microscopy, electron tomography, matlab, myosin, smooth muscle, structural biology / Includes bibliographical references. / Kenneth A. Taylor, Professor Directing Dissertation; P. Bryant Chase, Committee Member; Thomas C. S. Keller, Committee Member; Hong Li, Committee Member.

The DNA in the eukaryotic genome is wrapped in 147--bp segments around an octamer of histone proteins to form the fundamental subunit of chromatin, the nucleosome. Nucleosomes regulate the access of proteins to DNA, thus regulating important DNA-templated events such as transcription, translation, recombination, and repair. In order to characterize the chromatin landscape in maize, we mapped nucleosome positions using micrococcal nuclease (MNase) to enrich for nucleosomal DNA. We mapped nucleosomes under a variety of experimental conditions and in different tissues. We identified an unexpected, nonuniform source of variation which we traced to the degree to which chromatin is digested with MNase. We exploited this property to identify nucleosomes in the maize genome that possessed unique biochemical traits as being hypersensitive or hyper-resistant to MNase digestion. These regions were associated with important biological processes, including gene expression levels, transcription-factor binding, and highly-conserved noncoding sequences. In addition, we found that these nucleosomes displayed tissue specificity, implicating this special type of chromatin feature in regulating gene expression under different cell physiologies. We extended this work to the human genome and made similar discoveries: hypersensitive nucleosomes were associated with gene expression levels and were enriched in important regulatory elements. We also found hyper-resistant nucleosomes to be highly-associated with paused RNA polymerase II, implicating these nucleosomes in regulating transcriptional elongation. Thus, our approach to chromatin profiling uncovers novel biochemical states of multicellular organisms that are likely important for transcription, differentiation, and cellular responses. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2014. / November 10, 2014. / chromatin, genomics, microarrays, MNase-seq, ngs, nucleosome / Includes bibliographical references. / Hank Bass, Professor Co-Directing Dissertation; Jonathan Dennis, Professor Co-Directing Dissertation; Jinfeng Zhang, University Representative; Brian Chadwick, Committee Member; Dave Gilbert, Committee Member.

Insect flight muscle (IFM) is the preferred model system for visualizing actin-myosin interactions due to its highly ordered lattice of actin and myosin filaments. Electron tomography (ET) of fast-frozen, actively contracting Lethocerus IFM has recently resulted in a model for the weak to strong transition in the myosin crossbridges that produce force (Wu et al., 2010). These myosin molecules consist of a motor domain (MD), a lever arm and a coiled-coil rod domain that forms the filament backbone. The MD and lever arm region together constitute the subfragment 1 (S1) domain. The MD contains the ATP catalytic site and the actin-binding site. The myosin lever arm contains the essential and regulatory light chain bound to a long alpha helix. The first 50 nm of the rod domain consists of the subfragment 2 (S2) region, which acts both as a linker and adapter to transmit force produced by the MD and is sufficient to form the myosin dimer. Atomic models of myosin heads remodeled to fit the cross-bridge density show a distinctly straightened appearance when compared to the crystal structures of myosin subfragment 1 (S1). This implies that there is an aspect of the structural changes that occurs in force production that has not been recognized in myosin crystal structures of various intermediates. A weak-to-strong binding transition involving an azimuthal reorientation of the myosin MD on actin could explain this observation provided that myosin's α-helical coiled-coil S2 domain emerged from the thick filament backbone at a particular location. Previous studies did not visualize the S2 domain in either the raw tomogram or in subvolume averages. Here we have used ET of IFM fibers in rigor, in which the filament lattice has been swollen in low ionic strength buffer, to view where S2 emerges from the thick filament backbone as a test of the weak to strong transition. The results show that the S2 origins of those rigor myosin heads bound to the target zone of active muscle originate from the same region of the thick filament as implied by the position of the S1/S2 junction observed in active muscle. This shows the myosin heads in clear agreement with the previously proposed weak to strong transition model. In order to visualize IFM by ET, crossbridge samples must be sectioned because they are otherwise too thick. Sample preparation methods include fixation and embedding followed by sectioning and staining. These preparation steps can potentially induce artifacts. The most notable sectioning artifacts are compression and shearing of the specimen. We examined ~80 nm thick transverse sections (cross-sections) cut with both a vibrating knife (sonic-knife) and static knife and explored different knife settings. We examined the mitigating effects of these sectioning parameters on both compression during cutting and shear distortions on the filaments as seen in tomograms of rigor muscle swollen in low ionic strength medium. Separate from specimen preparation challenges, data collection also presents with a set of artifacts. Mass loss in plastic sections in conventional ET can reduce section thickness by as much as 30%. We evaluated the benefit of collecting tilt series at -190°C with < 60 e⁻/Å2 total exposure, a value that is 50% of the dose typically used in cryo-ET for frozen hydrated specimen, in order to minimize radiation-induced mass loss that results in section thinning. Reducing the artifacts in our sample facilitated reconstruction of the IFM lattice making it possible to probe aspects of muscle contraction in greater detail than previously possible. ET is most useful for imaging biological structures in situ. A sample with reduced artifacts opens the door for better reconstructions. We used subvolume averages of both thin and thick filaments to reassemble the filament lattice with high signal-to-noise ratio averages. We used the improved samples with the intent of resolving the subfilament structures in the thick filament backbones as well as the subunit structure in the actin thin filaments. The information obtained from these averages provided two separate frames of reference for deciphering the relationship between crossbridge origins on the thick filament with crossbridge binding sites on actin. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2014. / October 27, 2014. / Actin, Insect Flight Muscle, Lethocerus, Myosin, Thick filament, Thin filament / Includes bibliographical references. / Kenneth A. Taylor, Professor Directing Dissertation; Timothy Logan, University Representative; P. Bryant Chase, Committee Member; Thomas C. S. Keller, Committee Member; Scott Stagg, Committee Member.

Diphtheria toxin repressor is a regulatory protein from Corynebacterium diphtheriae, the causal agent of Diphtheria. The diphtheria toxin repressor (DtxR) contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment that serves to stabilize the inactive state of the repressor. During activation of DtxR by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active repressor. In this study we investigate the dynamics of this intramolecular complex using backbone amide nuclear spin relaxation rates determined experimentally using NMR spectroscopy and computed from molecular dynamics trajectories. The SH3 domain in the unbound and bound states showed typical dynamics in that the secondary structures were fairly ordered with high generalized order parameters and low effective correlation times while residues in the loops connecting b-strands exhibited reduced generalized order parameters and required additional motional terms to adequately model the relaxation rates. Residues forming the Pr segment also exhibited low order parameters with internal rotational correlation times on the order of 0.6 – 1 ns. Further analysis showed that the SH3 domain was rich in ms motions while the Pr segment was rich in motions on the 100s ms timescale. Molecular dynamics trajectories of PrSH3 and SH3 indicated structural rearrangements that might contribute to the observed relaxation rates and, together with the observed relaxation rate data, suggested that the Pr segment exhibits a binding ↔ unbinding equilibrium. The intramolecular complex resisted any significant change in the binding affinity between the Pr segment and the SH3 domain due to mutations in the Pr segment. The results of this study provide key insights into the nature of the intramolecular complex and provide a better understanding of the biological role of the SH3 domain in regulating DtxR activity. / A Dissertation Submitted to the Department of Chemistry and Biochemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Fall Semester, 2007. / October 23, 2007. / NMR, SH3 domain, relaxation, Lipari-szabo / Includes bibliographical references. / Timothy M. Logan, Professor Directing Dissertation; Huan-Xian Zhou, Outside Committee Member; Hong Li, Committee Member; Oliver Steinbock, Committee Member.

The current treatment for hepatitis C virus (HCV) consists of a combination therapy of alpha interferon (IFN-alpha) and ribivirin (RBV). Due to IFN resistance and side effects, new classes of drugs are needed to combat HCV infection. Cyclosporine A (CsA), an immunosuppressive and anti-inflammatory drug, has been shown to suppress HCV via a mechanism independent of the IFN pathway. In order to study the mechanism of CsA action on HCV, CsA resistant strains of HCV subgenomic replicon were selected and characterized. Here we report that different levels of resistance can be seen in different replicons and that different sets of mutations are associated with the different levels of resistance. Several different single cell clones with varying levels of CsA resistance contained mutations in the nonstructural protein 5B (NS5B), the HCV-encoded polymerase. When engineered into wildtype replicon these mutations were sufficient to confer a certain degree of resistance, but not to the original levels of selected replicons. Furthermore, these mutations, both individually and in groups, were able to rescue the lethal phenotype of a point mutation in NS5B (P540A) that has been previously implicated in the blockade of cyclophilins binding. These results demonstrate that CsA exerts selective pressure on the HCV genome despite being known to act on a cellular protein and identify a major target of CsA-mediated inhibition of HCV replication. / A Thesis Submitted to the Department of Biological Science in Partial Fulfillment of the Requirements for the Degree of Master of Science. / Fall Semester, 2009. / August 19, 2009. / CsA, Cyclosporine, Hepatitis C Virus, HCV / Includes bibliographical references. / Hengli Tang, Professor Directing Thesis; Thomas C. S. Keller, III, Committee Member; Fanxiu Zhu, Committee Member.

Molecules containing the phosphate (O—PO32-) moiety are ubiquitous in biochemistry. Phosphoryl transfer reactions that break and form the O—P phosphoryl bond are central to biological processes as diverse as energy metabolism and signal transduction. As described by Westheimer, the utility of phosphates stem from their ability to be kinetically stable while thermodynamically unstable. This dissertation uses electronic structure theory to investigate, at an elementary chemical level, the thermodynamic and kinetic properties of phosphate esters in an attempt to answer the question, "Why nature chose phosphates?". Chapter 1 formulates the question to be answered. Chapter 2 provides the underlying theoretical background to the computational methods employed. In Chapter 3, the anomeric effect, a stereoelectronic effect is first identified as a contributor to the high-energy status of N-phosphoryl-guanidines using electronic structure methods. In Chapter 4 it is further found that the anomeric effect can contribute to the thermodynamic poise of a range of phosphates. Chapter 5 investigates the connection between phosphoryl transfer mechanisms and the anomeric effect. It is found that the anomeric effect promotes O—P bond cleavage and plays a dominant role in the dissociative mechanism of phosphoryl transfer. The impact of other stereoelectronic effects such as hyperconjugation upon the hydrogen bonding properties of phosphates is also examined in Chapter 6. Compelling evidence is obtained suggesting the role of the O—P bond weakening anomeric effect in discriminating phosphoryl transfer potentials and controlling reaction rates in a range of biologically important phosphoryl compounds. Strong correlations between phosphoryl transfer potentials, rates of reaction in solution, O—P bond weakening, and the magnitude of the n(O)→σ*(O—P) anomeric effect is shown. This dissertation articulates a fundamental property of phosphates that may provide an answer to the age old question of "Why nature chose phosphates". / A Dissertation Submitted to the Program in Molecular Biophysics in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Summer Semester, 2007. / June 11, 2007. / Phosphates, Anomeric Effect / Includes bibliographical references. / Michael S. Chapman, Professor Co-Directing Dissertation; W. Ross Ellington, Professor Co-Directing Dissertation; Robert L. Fulton, Outside Committee Member; Timothy A. Cross, Committee Member; Huan-Xiang Zhou, Committee Member.

Myosin activity and actomyosin interaction play key roles in normal and pathological cardiac muscle contraction. Factors that modulate actomyosin have the potential to exert changes in cardiac contraction that may be necessary for normal cardiac function, or could be beneficial for the treatment of pathological cardiac function. Here we describe an investigation into both intrinsic factors that may contribute to the normal role of actomyosin in muscle contraction, as well as extrinsic factors, in the form of drugs, compounds, or gene therapy, that have potential for therapeutic treatment of cardiac disease states due to possible modulations of myosin activity or actomyosin interaction. We found that changes in myosin isoform that sometimes accompany cardiac disease states alter the kinetics of muscle contraction without alteration to the calcium sensitivity of contractile parameters. We also found that there may exist an important protein interaction between the thin filament protein troponin and myosin during normal muscle contraction. This interaction could have negative implications if mutations at that interaction site alter normal contraction. We found that the anti-hypertrophic drug rapamycin does not exert its cardiac anti-hypertrophy via its direct effects on the myofilament proteins. This suggests that rapamycin's activity rather may control signaling pathways involved in cardiac contraction. We found that low, but supra-physiological dATP/ATP ratios enhanced cardiac contractility in vitro, presumably due to recruitment of large numbers of force-producing actomyosin cross-bridges and an apparent high affinity of dATP for myosin. We showed that there may exist an "optimal" intracellular [dATP] that has the potential to enhance cardiac contractility in living cardiac cells. These results suggested that methods for increasing [dATP] in living cardiac tissue should be explored. Clearly, it is important to understand and investigate factors that directly modulate myosin activity and actomyosin interaction in order to contribute to future treatments for pathological cardiac conditions. / A Dissertation Submitted to the Program in Molecular Biophysics in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Fall Semester, 2006. / October 13, 2006. / Inotrope, ATP, Cardiac Muscle, Actin, Myosin / Includes bibliographical references. / P. Bryant Chase, Professor Directing Dissertation; Oliver Steinbock, Outside Committee Member; Kenneth Taylor, Committee Member; Timothy Moerland, Committee Member.