H-Ras is a member of the small, monomeric GTPase protein superfamily. H-Ras functions as a ?molecular switch?, using nucleotide dependent conformational changes to relay signals in a number of signal transduction pathways. Mutations in codons 12, 13 and 61 creates an oncogenic version of the protein which does not hydrolyze GTP, resulting in the constitutive activation of downstream effector proteins. Ras proteins participate in multiple protein : protein interactions in the cell, making Ras a good candidate protein to extend the Multiple Solvent Crystal Structures method (MSCS) to the analysis and prediction of protein binding surfaces. MSCS involves solving the crystal structure of the protein after soaking the protein crystal in a variety of organic solvent molecules. Replacing an aqueous solvent with an organic solvent affects the Ras protein structure in several ways. The disordered Switch II region of Ras is ordered in the presence of 2,2,2-trifluoroethanol or 1,6-hexanediol. Polar interactions that stabilize the ordered switch are enhanced in the presence of hydrophobic co-solvents. This suggests that hydrophobic solvents can be used in general to order short biologically relevant segments of disordered regions in protein crystals. We have used MSCS to study two crystal forms of active H-Ras bound to a nonhydrolyzable GTP analog (GMPPNP). We have also solved the structure of an oncogenic mutant of H-Ras (Q61L) in a non-canonical crystal form. This crystal form of H-Ras shows a new conformation for the flexible Switch II region that is not affected by crystal packing forces. This provides a structural explanation for the oncogenic properties of the Q61L mutation, showing that the Q61L mutation stabilizes a non-catalytic conformation of Switch II. MSCS analysis of Ras identifies the known Ras-effector binding domain as a site of protein: protein interaction and predicts a new protein binding site that is located in a large, solvent exposed pocket between Switch II and helix 3. In applying MSCS to the Ras protein, we show that by using polar organic solvent molecules as probes, we can identify binding sites that are highly charged and dynamic.

A complete understanding of protein synthesis requires description of the cyclical motions and interactions that occur in ribosomal RNA and transfer RNA during translation. The purpose of the following research was to gain understanding of the nature of these motions and interactions, and to develop new tools to measure how ligands alter the conformational flexibility of RNA during the steps of initiation, elongation and termination. Different tRNA substrates were bound to the E. coli 70S to simulate the arrangement of the tRNA-ribosomal complex before and after peptide bond formation, and different UV-induced 16S rRNA-tRNA photocrosslinks were produced in these complexes, illustrating that the 16S rRNA P-site undergoes local deformations during elongation. A statistical study was undertaken to understand the nature of conformational states in the 30S ribosomal subunit. Using the lists of observed UVB/C- and UVA-s4U-induced crosslinks and the T. thermophilus 30S X-ray crystal structure, frequencies were compared to a number of geometrical parameters demonstrating that crosslink formation requires substantial RNA motions. In addition, the results show that the restricted pattern of crosslink formation in E. coli 16S rRNA is due to the overall rigidity of the 30S subunit outside of the active site. One consequence of these conclusions is that potocrosslinking rates depend on the ease of inter-nucleotide conformational movements. This was exploited in a study that used the temperature response of the rate constant for the UVA-induced photo-crosslink between s4U8 X C13 in E. coli tRNA to determine tRNA geometry and internal energy. The rate constants followed Arrhenius behavior in their dependence on temperature, and this allowed calculation of the activation energy associated with the conformational rearrangement necessary to bring the photoreactive bonds together. The experiments show that changes in the tRNA on ribosomes can be uncovered by photocrosslinking, that RNA mobility occurs by transient conformational changes, and describe a new technique than can quantitatively measure the internal energy associated with these conformational movements.

The ribosome is a complex RNA-protein machine that translates the genetic information contained in the mRNA molecule to a sequence of amino acids. This involves mediating the decoding of the mRNA sequence, peptide bond formation and translocation. Throughout the process of translation, the ribosome undergoes significant conformational changes. The crystal structures of ribosome and the ribosomal subunits have been resolved at moderate resolution, but the details of conformational changes at various steps of translation have just started to be understood. Both structural and biochemical techniques have been applied towards the understanding of the ribosome dynamics. UVB (far UV, 210-300 nm) radiation has been used to study the structural changes in 16S rRNA within the 30S ribosomal subunit by monitoring the changes in the crosslinking frequencies of RNA-RNA crosslinks. In the first part of this study additional RNA crosslinks in 16S rRNA are determined after <i>in vivo</i> incorporation of 4-thiouridine (s⁴U) into RNA in a strain of <i>Escherichia coli</i> deficient in pyrimidine synthesis followed by irradiation with UVA (near UV, 300-380 nm) light. Similar crosslinks are observed when synthetic 16S rRNA transcribed with s⁴U is reconstituted into 30S subunits except that there are a few conformational differences between in vivo and in vitro synthesized subunits. Crosslinks obtained from UVB and s⁴U-UVA irradiation show a striking similarity between their identity and location. All the crosslinks are found to be restricted in the 30S subunit making a reversed ?C? shape through the center of the subunit. These results predict local RNA conformational flexibility that is most likely the factor in determining the formation of the crosslinks. In the second part, the effects of initiation factors on the structural status of the 30S initiation complex are studied. The results indicate IF3 is the main player in determining the 30S structure during initiation and that its C-domain (IF3C) causes similar structural changes in the 16S rRNA.

Archaeal box C/D sRNAs guide the 2?-O-methylation of target nucleotides in both ribosomal and tRNAs. These small non-coding RNAs are characterized by conserved terminal box C/D and internal C?/D? RNA motifs. Each RNA motif binds three core proteins to establish individual RNP complexes that catalyze the site-specific 2?-O-methylation of target nucleotides. Specificity of nucleotide modification is determined by target RNA base pairing with complementary sRNA D or D? guide sequences. The fifth target nucleotide upstream from the D or D? box within the guide:target RNA is then methylated by the core proteins. In vitro assembly of Methanocaldococcus jannaschii sR8 box C/D RNA with recombinant core proteins, L7, Nop56/58, and fibrillarin produces a methylation-competent sRNP complex. This model box C/D sRNP has now been used to determine the structural features of the guide:target RNA duplex that are important for sRNA-guided nucleotide methylation. Watson-Crick pairing of guide and target nucleotides was essential for nucleotide methylation. Mismatched bases within the guide:target RNA duplex also disrupted target nucleotide methylation. Nucleotide methylation required that the guide:target duplex consist of an RNA:RNA helix as target deoxy-oligonucleotides possessing a target ribonucleotide were not methylated. Methylation specificity at the base paired guide:target nucleotide was compromised by elevated Mg2+ concentrations. In high divalent cation concentrations, target nucleotides not hydrogen bonded to the guide nucleotide were nevertheless methylated. Interestingly, D and D? target RNAs were methylated to different levels when deoxynucleotides were inserted within the target RNA or when target methylation was carried out in elevated Mg2+ concentrations. These results suggested that structural features unique to the box C/D and C?/D? RNPs affect their nucleotide methylation capabilities. Finally, the ability of the sR8 box C/D sRNP to methylate target nucleotides positioned within highly structured RNA hairpins suggested an intrinsic ability of this archaeal RNA:protein enzyme to unwind double-stranded target RNAs prior to nucleotide modification.

Follicle stimulating hormone (FSH) is a a/b glycoprotein produced in pituitary gonadotropes of all vertebrates and is required for egg maturation, and optimal sperm performance. Activin potently induces FSH by inducing transcription of its rate-limiting b-subunit (FSHB). Four nucleotides of the oFSHB promoter (â168 bp to â165 bp) are necessary for 99.9 % of oFSHB expression in vivo and 70 % of its progressive induction by activin in LbT2 gonadotropes over a 24 hr period. These 4 nucleotides form part of a putative forkhead box (FOX) binding site juxtaposed upstream to a single-copy (4 bp) Smad binding element (SBE), both of which are associated with activin action. Smad4 from LbT2 cells did not bind the wild type oFSHB SBE in electrophoretic mobility shift assays, but did bind a palindromic SBE derived from the oFSHB SBE. Binding increased 2.6-fold over 20 h without or with activin (25 % additional increase with activin) and was competed 85 % with the native oFSHB sequence indicating Smad4 has high affinity for the native promoter. Additionally, a dominant negative inhibitor of Smad4 reduced activin induction of oFSHB in LBT2 cells by 62 %, indicating that Smad4 is important for activin induction. A second Smad, Smad3, bound transiently to the palindromic SBE (6-fold increase by activin at 2 h). Dominant negative inhibition of Smad3 (3SA) and depletion of Smad2 by siRNA did not alter activin induction of oFSHB suggesting Smads 2 and 3 may not be involved. A p38 inhibitor blocked induction of oFSHB after 8 h, dividing the 24 hr induction by activin into two phases. This suggested that an activin-regulated early gene product is required for the second phase of oFSHB induction. It was found that one forkhead gene, FOXQ1, was increased 4.5-fold 8 h after activin treatment which correlated nicely with the second phase of oFSHB induction uncovered by the p38 inhibitor. FOXQ1 is only one of 43 FOX family members, however, so further studies are required to prove that FOXQ1 is a key driver of FSH production and that it partners with Smad4 to induce oFSHB transcription.

Bacteria remarkably and constantly adapt to their surrounding environment, especially in times of considerable environmental stress. These responses range from secretion of toxins, antibiotics and complete physiological transformations leading to the development of highly resilient spores resistant to heat, sunlight, chemicals and drugs. Due to the constant flux in environmental conditions, bacteria spend most of its life in this critical period, the transition-state. Proteins involved in the activation of genes required for survival are called transition-state regulators, a novel family of DNA binding proteins. Two defining characteristics describe transition-state regulator proteins: 1.) ability to recognize a multitude of genes with no consensus sequence and 2.) sole decision making policy of the cell is controlled by this select group proteins to monitor and regulate hundreds of cellular pathways. Despite their vital and increasingly common role, there is a paucity of information. <i>Bacillus subtilis</i> and <i>B. anthracis</i> both possess transition-state regulators responsible for gene expression during transition from vegetative to post-exponential growth. This dissertation focuses on investigating the protein-DNA interactions of the transition state regulators, antibiotic resistance protein B (AbrB) and antibiotic resistance protein h (Abh) from <i>B. subtilis</i> and <i>B. anthracis</i> to develop a detailed general model describing recognition and interaction mechanisms. Biophysical characterization of AbrBN and AbhN show that multimerization plays an essential role in binding to DNA targets. High-resolution NMR structure analysis was carried out to refine the structure of AbrBN and to solve the structure of AbhN, both showing dimeric topologies. These studies employed a complementary mutagenesis, ESI-MS, NMR, as well as a multiple spectroscopic technique approach. The study concluded with a respectable model of AbrB?s, and transition state regulators in general, interaction with DNA. The unique ability to bind over 60 genes with no consensus sequence relies on conformational flexibility of both protein and DNA. Furthermore, the solution structure of the N-terminal domain of Abh provided the stature needed to declare the transition-state regulator family a unique and characterized DNA binding family.

Caspases are a family of cysteine proteases that are intimately involved in apoptosis and exist in the cell as inactive zymogens prior to activation. Initiator procaspases are monomers that must dimerize for activation. Executioner procaspases, such as procaspase-3, are dimers that must be processed for activation. The chemical properties of the dimer interface are different between the two subfamilies of caspases but their structures are similar, suggesting that the interface region is important for regulation. The goal of the studies presented here is to determine the importance of the dimer interface in folding and assembly of procaspase-3. A histidine mutation was introduced into the dimer interface region, which completely abolished the activity of mature caspase-3. Equilibrium and kinetic folding studies were performed to elucidate how a mutation in the dimer interface prevents substrate turnover in the active site when the distance between the two regions is 20Ã. The folding studies presented here coupled with the crystal structure show that the protein is entering a kinetic trap prior to dimerization because the histidine has to adopt an unusual rotomer to pack into a region that normally accommodates a much smaller valine residue. A hysteresis was observed by equilibrium folding studies, presumably because the time it takes to refold is different that the time it takes to unfold due to the limited conformational freedom of the histidine residues. Additionally, the hysteresis is observed to be concentration dependent suggesting that the histidine residue makes the activation mechanism of procaspase-3 more like initiator caspases that require a large local concentration of protein to promote dimerization. Kinetic refolding studies showed that the procaspase-3 monomer is becoming trapped in a conformation that is unstable and prone to aggregation prior to forming a dimerization competent species. The crystal structure of caspase-3 (V266H) revealed two separate pathways of inhibition starting from the dimer interface and culminating in the active site that could be responsible for the lack of activity in this mutant. These data, taken together, suggest that the dimer interface is a region that can be used to allosterically inhibit procaspase-3 because it is important for regulation of the enzyme. This is important because it could be used as a drug target for diseases that have too much cell death, such as neurodegenerative disorders.

The ability of bacteria to adapt to environmental stress is essential to their existence. Several responses to environmental triggers are known. Two of these, Competence and Sporulation, are widespread in the bacterial world. Competence and Sporulation are best studied in the organism Bacillus subtilis and the proteins involved in these pathways are well known. The purpose of this research is to gain insight into the regulation of these cellular processes by determining the NMR solution structures and looking at functional aspects of the ClpC R-domain, involved in bacterial competence, and the L66A mutant of the Spo0F protein involved in the regulation of sporulation. In addition to these studies attempts to purify a Spo0F homolog from the endospore-forming bacteria, Pasteuria penetrans, were made with the intention of determining the metal binding characteristics of the protein.

Geminiviruses are small DNA viruses that replicate in the nucleus and use plant replication machinery to amplify their single-stranded genomes. Geminiviruses replicate through a combination of rolling-circle and recombination-dependent replication. Earlier studies showed that the geminivirus, Cabbage leaf curl virus (CaLCuV), induces transcription of a host gene encoding a replication factor. To scrutinize the global impact of geminivirus infection on host gene expression, Arabidopsis transcriptome in response to CaLCuV infection at 12 days post inoculation was examined. These experiments uncovered 5241 Arabidopsis genes with changes in transcript levels (q < 0.004, p < 0.002) in response to infection. Data mining of the differentially expressed genes revealed that CaLCuV triggers a pathogen response via the salicylic acid pathway and inhibits the jasmonate pathway. CaLCuV also induced genes associated with programmed cell death and genotoxic stress, including components of the host DNA repair apparatus. CaLCuV also impacted expression of cell cycle-associated genes, preferentially activating genes with peak expression in S and G2 and inhibiting genes that peak in G1 and M. A limited set of core cell cycle genes expressed during cell cycle re-entry, late G1, S and early G2 had increased RNA levels, while core cell cycle genes expressed in early G1 and late G2 had reduced transcript levels. FACS analysis of nuclei from infected leaves showed an increase in 8C, 16C and 32C DNA content relative to nuclei from control leaves. Together, these results underscore the complexity of geminivirus/host interactions and establish that geminiviruses alter cell cycle controls to induce plant cells to move into an endocycle and support viral replication. We also standardized a previously reported method for inoculating geminiviruses using plasmid DNA rubbed onto leaves in the presence of an abrasive (DNA abrasion). We can obtain 100% inoculation efficiency with this method in solanaceous plants. Silencing of an endogenous gene was successful when a silencing vector was inoculated by DNA abrasion.

We have examined the folding, assembly and active site conformation of a catalytically inactive mutant of procaspase-3 (procaspase-3(C163S)), a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. We show that an uncleavable mutant (D3A) of procaspase-3 is catalytically active with Km similar to that of its mature counterpart. We developed limited proteolysis assays using trypsin and V8 proteases, which allow the examination of amino acids in three of five active site loops. In addition, we examined the response of the two tryptophanyl residues in the active site to several quenching agents over the pH range of 3 to 9. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle between loops L4, L2 and L2?. The pKa?s of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate-binding pocket, does not appear to be unraveled and solvent exposed, even at lower pH. In order to understand the active site formation in the context of folding, we looked into the equilibrium unfolding of procaspase-3(C163S). The equilibrium unfolding of procaspase-3(C163S) is described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. Therefore, dimerization is a folding event and it contributes significantly to the protein stability (18.8 kcal/mol of 25.8 kcal/mol). Equilibrium unfolding experiments of procaspase-3(C163S) at different pH shows maximum stability at pH 7.2, and a transition from four-state model to a three-state monomer and finally to a two-state monomer model with decrease in pH. This is representative of conformational change and dimer dissociation at lower pH (between pH 5 and 4). The pro-less variant of procaspase-3(C163S) folds reversibly only in 1:1 protein to pro-peptide ratio suggesting it might act as an intramolecular chaperone (IMC).