Dissertations / Theses on the topic 'General biochemistry'

Calmodulin (CaM) plays an important role in calcium (Ca²⁺)-dependent signal transduction. Ca²⁺ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca²⁺-dependent inactivation (CDI) process in store-operated Ca²⁺ entry (SOCE), by interacting with the N-terminus of the hexameric plasma membrane Ca²⁺ channel Orai1. To understand the relationship between Ca²⁺-induced hydrophobicity of CaM and the CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) were used as an informative probe to better understand the functionality of each EF-hand. ANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca²⁺ binding. The exchanged EF-hands from TnC to CaM resulted in reduced hydrophobicity compared with wild-type CaM, as depicted by ANS fluorescence and binding affinity. Such a conclusion is consistent with general concepts about the inadequacy of hydrophobic exposure for chimeras. However, such ANS responses exhibited no correlation with the ability to interact with Orai1. ANS lifetime measurements indicated that there are two types of ANS molecules with rather distinct fluorescence lifetimes, each specifically corresponding to one lobe of CaM or chimeras. Thermodynamic studies indicated the interaction between CaM and a 24-residue peptide corresponding to the CaM-binding domain of Orail1 (Orai-CMBD) is a 1:2 CaM/Orai-CMBD binding, in which each peptide binding yields a similar enthalpy change (ΔH = − 5.02 ± 0.13 kcal/mol) and binding affinity (K_a = 8.92 ± 1.03 x 105 M⁻¹). With the exchanged EF1 and EF2, the resulting chimeras noted as CaM(1TnC) and CaM(2TnC), displayed a two sequential binding mode with a one-order weaker binding affinity and lower ?H than that of CaM, while CaM(3TnC) and CaM(4TnC) had similar binding thermodynamics as CaM. Circular Dichroism studies suggested differences in binding most likely resulted from changes in chimera three-dimensional structure rather than secondary structure, as the extent of ?-helical content from apo-, Ca²⁺-, and Orai-CMBD-bound proteins remained similar. The dissociation rate constant for CaM/Orai-CMBD was determined to be 1.41 ± 0.08 s⁻¹ by rapid kinetics. Stern-Volmer plots of Orai-CMBD Trp76, indicated that the residue is located in a very hydrophobic environment but becomes more solvent accessible when EF1 and EF2 were exchanged. Here, the model of 1:2 binding stoichiometry of CaM/Orai-CMBD established in solution supports the unique, open binding mode suggested by already published structural studies.

Surface plasmon resonance (SPR) active nanohole array substrates offer a diverse biosensing platform with high sensitivity and unique characteristics. This dissertation investigates the sensitivity and fundamental SP features of various nanohole array substrates and demonstrates higher sensitivity than conventional continuous gold platforms, tunability to specific analytes, and great enhancement of the local field intensity. Novel instrumentation and analytical techniques are developed and utilized to assess the nanohole array SPR sensing substrates in the near infrared as well as with interaction of other nanostructures.

The nanohole array substrates are evaluated throughout the near-infrared (NIR) region by novel SPR instrumentation and methodology that extends the working SPR wavelength range and measurement reliability. Development of a robust NIR-SPR instrument allows access to higher wavelength ranges where sensitivity is improved and novel SP modes and plasmonic materials may be investigated. Different aspects of the NIR-SPR instrument, including temporal stability, mechanical resilience and sensitivity, are evaluated and presented. Furthermore, a method is developed for improving precision and accuracy of empirically determined SP penetration depth, a merit of SPR spectroscopy sensitivity. The technique incorporates an adsorbate-metal bonding effect which improves the consistency in the penetration depth value calculated at different adsorbate thicknesses from 41-1089% relative deviation (without bonding effect) to 2-11% relative deviation (with bonding effect). It also improves the experimental agreement with theory, increases the accuracy of assessing novel plasmonic materials and nanostructures, and increases the precision in adsorbate parameters calculated from the penetration depth value, such as thickness, binding affinity, and surface coverage.

Utilizing this NIR-SPR instrument and improved technique for calculation of penetration depth, the sensitivity and various SP modes of the nanohole arrays throughout the NIR range are evaluated, and an improvement in sensitivity compared to conventional continuous gold is observed. Both the Bragg SPs arising from diffraction by the periodic holes and the traditional propagating SPs are characterized with emphasis on sensing capability of the propagating SPs. There are numerous studies on the transmission spectroscopy of nanohole arrays; however this dissertation presents one of the few studies in Kretschmann mode, and the first in the near infrared, where greater surface sensitivity is observed. The sensitivity profile of various nanohole array parameters (periodicity, diameter, excitation wavelength) and SP modes is also presented.

Further control and enhancement of the SP field is pursued by interaction between nanohole array substrate and nanoparticles to exploit field intensification between plasmonic structures, i.e. gap mode enhancement. Under specific conditions, the SPs couple together and the electric field between the structures is amplified and localized, which may be exploited for sensing purposes and surface enhanced techniques, including tip enhanced Raman spectroscopy (TERS) or surface enhanced Raman spectroscopy (SERS). A technique for observing nanohole array-nanoparticle distance dependent SP interaction is developed and utilized to demonstrate SP interaction. Scanning probe microscopy controls the position of a single nanoparticle (SNP) affixed to an atomic force microscope probe, and the location specific interaction of the SNP-nanohole array surface plasmons is measured by darkfield surface plasmon resonance spectroscopy. Coupling of the nanoparticle to the nanohole array exhibits a maximum when the SNP resides within a nanohole, which resulted in a maximum SPR wavelength shift of 17 nm and an increase in scatter intensity. This dissertation presents the first empirical observations of SPM controlled gap mode enhancement of more complex nanostructures and allows for optimization of positioning prior to use in sensing.

The interplay between sequence, structure and function is an underlying theme in biological systems. Proteins, in particular, have evolved the ability to access a virtually infinite set of three-dimensional architectures from a small collection of building blocks; it is precisely this complexity of form that finely tunes their functional specificity. β-Peptides are a class of unnatural polyamides known to adopt structural motifs that are in many ways reminiscent of protein folds in nature. This dissertation first investigates the relationship between sequence and structure in self-assembling β-peptides, then demonstrates how the latter translates into function.

Chapter 1 provides an overview of the fundamental principles guiding β-peptide helix formation and self-assembly, and describes their applications both within and outside of the biological context. The ability of β-peptides to mimic natural α-helices while maintaining proteolytic resistance allows them to serve as therapeutic agents by targeting, for example, protein-protein interactions. Their unique stability in both aqueous and organic environments further enables the development of β-peptide-based nanomaterials and organocatalysts.

Chapter 2 elucidates the relationship between β-peptide primary sequence and quaternary structure based on the biophysical characterization of the Acid-3Y bundle. Acid-3Y was designed by substituting isoleucine for leucine side-chains in the sequence of the previously characterized octamer, Acid-1Y. The finding that Acid-3Y assembles into a tetrameric bundle suggests that branching at the γ-carbon of hydrophobic residues plays a critical role in determining β-peptide bundle stoichiometry.

Chapter 3 explores the potential of β-peptide bundles to mimic enzyme structure and function. The demonstration of β-peptide mutarotase activity in benzene highlights the importance of macromolecular preorganization in catalysis, while the ability of rationally designed β-peptide bundles to catalyze ester hydrolysis in water represents a crucial step towards the functionalization of these unnatural macromolecules. The dependence of catalytic activity on both active site geometry and bundle assembly, together with their substrate selectivity, underscores the unique biomimetic capacity of β-peptides.

Chapter 4 describes the rational design of a β-peptide ligand for the parathyroid hormone 1 receptor (PTH1R). Using previous strategies that led to the identification of p53 and GLP-1 mimics, a 12-member β-peptide library was constructed and tested in vitro for binding to the receptor protein. Although no hits were found from this initial screen, subsequently designed α/β-peptide chimeras showed promise as synthetic antagonists of PTH1R with improved pharmacokinetic properties.

Chapter 5 summarizes the key results of this dissertation and offers a perspective on possible future research directions. A breakthrough in the field of β-peptides would rely on the development of a method to synthesize genuine "β-proteins" with more sophisticated structure and function.

Electron cryomicroscopy (cryo-EM) techniques allow for the determination of cellular structures in three dimensions. The single-particle reconstruction (SPR) technique uses images of biological macromolecular complexes in a thin vitreous ice layer to generate three-dimensional (3D) reconstructions of them, and is capable of yielding structures of complexes ranging in size from hundreds of kilodaltons to tens of megadaltons.

Beam-induced motion (BIM) is one of the major causes of a decrease in the signal-to-noise ratio (SNR) in images obtained in electron cryomicroscopy. BIM is shown to be due to a physical doming movement of the ice sheet which can lead to translations of particles of up to several nanometers in the image plane. We show that computational correction of BIM using movie data can generate high resolution reconstructions with a fraction of the images previously required. SPR density maps also suffer from artifacts if images averaged into reconstructions represent a heterogeneous mixture. An algorithm capable of rapid classification of heterogeneous image data is presented and characterized. The algorithm is capable of similar performance as previously characterized algorithms.

The ribosome synthesizes mRNA encoded polypeptides. During its catalytic cycle, after peptidyl transfer, tRNA must move through the ribosome from the A and P sites to the P and E sites, respectively, in a process called translocation. This activity is accelerated 1000-fold by the GTPase EF-G. The structure of a transient early translocation intermediate bound with EF-G was determined using the new classification algorithm. The structure provides insight into the conformational changes that occur on the ribosome, tRNA and EF-G during translocation. Ribosomal cap-dependent initiation is a tightly regulated process directing initiation of polypeptide synthesis in eukaryotes. Internal ribosomal entry sites (IRESs) are RNA structures which allow the translation of downstream encoded elements to bypass some or all of the molecular machinery in cap-dependent initiation. The structure of the TSV IRES bound to the yeast ribosome is described. It shows that the IRES tRNA mimic is bound at the A site, and must undergo two rounds of translation prior to the formation of the first peptide bond.

Membrane proteins represent an important class of proteins that closely associate or reside within the plasma membrane of the cell. They play a multitude of roles in cell function such as signaling, trafficking, and recently discovered, scaffolding and shaping of the plasma membrane itself. For example, caveolin is a membrane protein that is believed to have the ability to curve the plasma membrane forming invaginations that serve as signaling platforms called caveolae. The curvature of the plasma membrane is believed to be a result of caveolin oligomerization. Caveolin oligomerization was characterized using sedimentation equilibrium analytical ultracentrifugation. Due to the extremely hydrophobic nature of caveolin it was necessary to explore different detergents and lipid systems that support membrane protein structure and function. Not all detergents are conducive to studies of membrane proteins and it is often necessary to determine empirically the best detergent / lipid mimic best suited for biophysical studies. One membrane mimic that has been well-characterized and used successfully to study membrane proteins are bicelles. Bicelles are discoidal phospholipid structures comprised of a long-chain and short-chain phospholipid, typically 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DHPC), respectively. Bicelles provide a true bilayer environment in which to study membrane protein structure and function. These lipid structures were successfully density matched using the method of sedimentation equilibrium in the analytical ultracentrifuge by adding 71.7% D₂O as a density modifier. We explored the utility of bicelles as a medium for studying membrane protein interactions in the analytical ultracentrifuge (AUC) by investigating the interactions of caveolin-1. The results of this work show that caveolin-1 does not have the capacity to oligomerize in detergent micelles or in a bilayer environment (bicelles). On the other hand, a naturally-occuring breast cancer mutant, P132L, forms a strong dimer in detergent micelles. A close investigation of the mutant reveals that an extension of helix 2 in the intramembrane region of the protein where dimerization was shown to occur may play a key role in the dimerization of the mutant.

An alternative bicelle system was also investigated using pentaethylene glycol monooctyl ether (C₈E₅) instead of DHPC to form the rim of the bicelle. The C₈E₅ / DMPC lipid aggregates were density matched and their properties were characterized using ³¹P-phosphorus NMR to assess the heterogeneity of the lipid / detergent arrangement, which confirms a bicellar-like arrangement. C₈E ₅ has a density similar to water (1.007 g / mL) and was shown to form lipid aggregate structures with DMPC that are less dense and require significantly lower quantity of D₂O to density match in the AUC making them better suited to the study of membrane protein interactions of small peptides.

This work describes the fundamental study of two bacterial toxins with computational methods, the rational design of a potent inhibitor using molecular dynamics, as well as the development of two bioinformatic methods for mining genomic data. Clostridium difficile is an opportunistic bacillus which produces two large glucosylating toxins. These toxins, TcdA and TcdB cause severe intestinal damage. As Clostridium difficile harbors considerable antibiotic resistance, one treatment strategy is to prevent the tissue damage that the toxins cause. The catalytic glucosyltransferase domain of TcdA and TcdB was studied using molecular dynamics in the presence of both a protein-protein binding partner and several substrates. These experiments were combined with lead optimization techniques to create a potent irreversible inhibitor which protects 95% of cells in vitro. Dynamics studies on a TcdB cysteine protease domain were performed to an allosteric communication pathway. Comparative analysis of the static and dynamic properties of the TcdA and TcdB glucosyltransferase domains were carried out to determine the basis for the differential lethality of these toxins. Large scale biological data is readily available in the post-genomic era, but it can be difficult to effectively use that data. Two bioinformatics methods were developed to process whole-genome data. Software was developed to return all genes containing a motif in single genome. This provides a list of genes which may be within the same regulatory network or targeted by a specific DNA binding factor. A second bioinformatic method was created to link the data from genome-wide association studies (GWAS) to specific genes. GWAS studies are frequently subjected to statistical analysis, but mutations are rarely investigated structurally. HyDn-SNP-S allows a researcher to find mutations in a gene that correlate to a GWAS studied phenotype. Across human DNA polymerases, this resulted in strongly predictive haplotypes for breast and prostate cancer. Molecular dynamics applied to DNA Polymerase Lambda suggested a structural explanation for the decrease in polymerase fidelity with that mutant. When applied to Histone Deacetylases, mutations were found that alter substrate binding, and post-translational modification.

A promising renewable energy scenario involves utilizing microalgae as biological solar cells to capture the energy in sunlight and then harvesting the biomass for renewable energy production. Through photosynthesis photons are captured by light-sensitive pigment molecules and used to create a cellular chemical energy gradient. Microalgae ultimately use this energy gradient to drive their metabolism by reducing inorganic carbon into renewable, energy-rich organic hydrocarbon stores such as triacylglycerols (TAGs). These valuable molecules act as a cellular energy reserve, readily drawn from when required, often forming large oil-bodies within microalgal cells that can be abundant in certain oleaginous species. This is important for biofuel production because lipids can be extracted from biomass and then converted into a variety of biofuels such as renewable diesel and jet fuel. Thus, from a biofuels perspective, maximizing lipid productivity in selected microalgal feedstock strains is considered essential to the development of an economically viable algal biofuels industry. To achieve this, many current research and development efforts are directed towards genetically engineering well-characterized microalgae to optimize TAG production; however, this approach is a time-consuming, costly prospect and the number of well-characterized strains is relatively few, especially when compared to the number of known extant species. Alternatively, microalgal feedstock optimization could be more readily accomplished by taking advantage of the prodigious natural diversity of microalgae in the environment and identifying native strains of microalgae that, through natural selection, already possess key metabolic traits necessary for commercial feedstock development. Formulated on this premise, a collaborative project between the National Renewable Energy Laboratory (NREL) and the Colorado School of Mines (CSM) recently established and cryopreserved a clonal microalgal culture collection containing 360 unique strains with preliminary data regarding lipid accumulation and the growth potential of select isolates. The goal of this work has been to 1) perform a far more detailed characterization of the algal culture collection by developing high throughput screening procedures and tools for identifying fast-growing, oleaginous strains; and 2) gather further insight into the microalgal diversity found in the southwestern United States. Herein is described in detail the rationale, methods, results and conclusions of these efforts.

Base stacking provides stability to nucleic acid duplexes, and base unstacking is involved in numerous biological functions related to nucleic acids, including replication, repair, transcription, and translation. The patterns of base stacking and unstacking in available nucleic acid crystal structures were classified after separation into their individual single strand dinucleotide components and clustering using a k-means-based ensemble clustering method. The A- and B-form proximity of these dinucleotide structures were assessed to discover that RNA dinucleotides can approach B-form-like structures. Umbrella sampling molecular dynamics simulations were used to obtain the potential of mean force profiles for base unstacking at 5'-termini for all 16 dinucleotides. A rate calculation method was investigated and implemented using small test compounds and applied to a base unstacking transition to predict a rate for 5'-terminal base fraying. The findings can be applied for localized nucleic acid structure prediction, and for comparison of molecular dynamics simulation-based investigations of nucleic acid distortions to experimental structural data.

In the D1-D61N mutant, it was possible to resolve a clear lag phase prior to the appearance of O2, indicating formation of an intermediate before onset of O2 formation. The lag phase and the photochemical miss factor were more sensitive to isotope substitution in the mutant indicating that proton efflux in the mutant proceeds via an alternative pathway. The results are discussed in comparison with earlier results obtained from the substitution of CP43-Arg357 with lysine and in regards to hypotheses concerning the nature of the final steps in photosynthetic water oxidation. These considerations lead to the conclusion that proton expulsion during the initial phase of the S3-S0 transition starts with the deprotonation of primary catalytic base, probably CP43-Arg357, followed by efficient proton egress involving the carboxyl group of D1-D61 in a process that constitutes the lag phase immediately prior to O2 formation chemistry. The asparagine, phenylalanine and threonine substitutions to D1-V185 were able to accumulate significant levels of charge separating PSII. Of the three substitutions the phenylalanine substitution was the most severe with a complete inability to evolve oxygen, despite being able to accumulate Photosystem II and to undergo stable charge separations. The threonine substitution had no apparent effect on oxygen evolution other than a 40% reduction in the steady state rate of O2 production compared to type Synechocystis, which can be attributed to that mutants reduced ability to accumulate PSII. The asparagine substitution produced the most complex phenotype. While still able to evolve oxygen, it does so less efficiently than wild type PSII, with a miss factor 4% higher than wild type Synechocystis. The substitution on D1-Val185 with asparagine also decreased the t1/2 of O2 release from thylakoid membranes from 1.2 ms to 10.0 ms and decreased the t1/2 lag phase prior to the onset of O2 release to 2.8 ms. The combination of a long lag period and a decreased rate of O2 release can also be observed in the D1-D61N mutant strains of Synechocystis and in PSII centers in which chloride has been replaced by iodide.

The cytoplasmic ring (C-ring) of the flagellar motor consists of three proteins: FliG, FliM, and FliN, each present in different copy numbers. These proteins perform the function of transmitting torque from the stators to the basal body, as well as regulating the rotational direction of the flagellum. Despite decades of study and great progress towards the understanding of the molecular details of the flagellum’s mode of action, substantial questions still remain about its detailed architecture and molecular mechanisms. Here we describe a series of in vitro and in vivo experiments designed to provide insight into the structure of the flagellar C-ring.

We begin this work by presenting a background of the forms of cellular motility and provide context for the flagellum within the great diversity of motility mechanisms. We then summarize the bacterial chemotaxis signal transduction system, one of the most deeply characterized signaling pathways within biology. Lastly we introduce the flagellum with a focus on C-ring structure and function.

The research portion begins with a characterization of the interaction between the FliG N-terminal domain (FliG_N) and the C-terminal region of the flagellar membrane protein FliF (FliF_C). We find that these two proteins interact strongly and that this interaction causes widespread conformational changes throughout FliG_N. Based on NMR and other biophysical data we propose a binding site for FliF_C centered on helix 1 of FliG_N.

In the next section we further characterize the interaction between FliF _C and FliG_N. We generate a fusion FliF_C-FliG _N polypeptide and characterize this complex. Using spin labeling experiments we confirm our predicted interaction site between FliF_C and FliG _N. We also identify a novel interaction between an important hydrophobic patch on the FliG_NM linker and FliG_N.

Next we study and characterize the domain architecture of the full-length FliG_NMC protein. By evaluating pair-wise domain interactions and comparing NMR spectra of numerous FliG constructs, in combination with in vivo experiments, we provide evidence that the FliG middle- (FliG_M) and C-terminal (FliG_C) domains interact in an intra-protomer manner. This model is in excellent accord with the 3D structure of the Salmonella typhimurium C-ring as derived from cryo-EM.

Lastly, we describe a number of experiments probing complex formation between FliG, FliM and FliN, with the aim of determining how these interactions are modulated by CheY binding.

Acidic aqueous-phase pretreatment is a promising approach that has been directed at maximizing intermediates yields (e.g. sugars, sugar degradation products, and lignin) from biomass for fuel and chemical production. This dissertation explores the kinetic fundamentals of biomass hydrolysis in acidic aqueous-phase with different catalysts (e.g. sulfuric acid, metal chlorides), operating conditions (e.g. temperature, time pressure), and equipment configurations (e.g. batch, flowthough).

The kinetic analysis revealed that crystalline cellulose is insusceptible to hydrolysis compared with agarose at low temperature (e.g.140 °C), while it decomposed rapidly at elevated temperature (e.g. 220 °C). Higher temperature with reduced time was desirable for glucose production whereas lower temperature with prolonged time was preferred for xylose generation. In acidic conditions, furfural and levulinic acid were stable whereas 5-hydroxymethylfurfural was susceptible to decomposition with high rate constant. MgCl₂ can promote the cleavage of C-O-C bond in polysaccharides (e.g. agarose) and enhance the subsequent dehydration reaction to 5-hydroxymethylfurfural. Unlike transition metal chlorides and H2SO₄, MgCl₂ has little ability to induce retro aldol and rehydration reactions to generate byproducts like lactic acid and levulinic acid. Mg²⁺ possessing hgiher activity than other alkali and alkaline earth metal chlorides (Na⁺ and Ca2⁺) resulted in 40.7% yield and 49.1% selectivity of 5-hydroxymethylfurfural.

Dissolution of biomass was significantly enhance using acidic hot water flowthrough pretreatment at 200—280°C. Significant cellulose removal accompanied with the transformation of cellulose I to cellulose II and amorphous cellulose were observed when temperature was above 240 °C for water-only and 220 °C for dilute acid. Approximately100% of the xylan and ∼90% of the cellulose were solubilized and recovered. Up to 15% of the lignin was solubilized, while the remaining lignin was insoluble. Over 90% sugar yields were obtained from pretreated whole slurries using less than 10 FPU/g cellulase plus hemicellulase enzyme.

A kinetic model was developed to depict the biomass degradation in flowthrough system. This model predicted the sugar generation more precisely than the conventional homogeneous first-order reaction models. Mass transfer limitations were minimized using 4mm biomass particle sizes with 4g biomass loading at 25mL/min flow rate, produced hydrolyzate slurries with 13g/L potential sugar concentrations.

Dicer plays a central role in RNA interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) or microRNAs (miRNAs), respectively. In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and either the RNA-binding protein TRBP or PACT to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs from Dicer to AGO2. Here, I have used electron microscopy and single particle analysis of human Dicer-RNA complexes and the RLC to gain insight into the structural basis for human Dicer's substrate preference and RISC-loading. My studies show that Dicer traps pre-siRNAs in a non-productive conformation, while interactions of Dicer with pre-miRNAs and dsRNA binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. The RLC Dicer's N-terminal DExH/D domain, located in a short base branch, interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.