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- All Subjects: Biophysics
- All Subjects: Photosynthesis
- Creators: Ros, Robert
- Creators: Fromme, Petra
Description
The need for a renewable and sustainable light-driven energy source is the motivation for this work, which utilizes a challenging, yet practical and attainable bio-inspired approach to develop an artificial oxygen evolving complex, which builds upon the principles of the natural water splitting mechanism in oxygenic photosynthesis. In this work, a stable framework consisting of a three-dimensional DNA tetrahedron has been used for the design of a bio-mimic of the Oxygen-Evolving Complex (OEC) found in natural Photosystem II (PSII). PSII is a large protein complex that evolves all the oxygen in the atmosphere, but it cannot be used directly in artificial systems, as the light reactions lead to damage of one of Photosystem II's core proteins, D1, which has to be replaced every half hour in the presence of sunlight. The final goal of the project aims to build the catalytic center of the OEC, including the Mn4CaCl metal cluster and its protein environment in the stable DNA framework of a tetrahedron, which can subsequently be connected to a photo-stable artificial reaction center that performs light-induced charge separation. Regions of the peptide sequences containing Mn4CaCl ligation sites are implemented in the design of the aOEC (artificial oxygen-evolving complex) and are attached to sites within the tetrahedron to facilitate assembly. Crystals of the tetrahedron have been obtained, and X-ray crystallography has been used for characterization. As a proof of concept, metal-binding peptides have been coupled to the DNA tetrahedron which allowed metal-containing porphyrins, specifically Fe(III) meso-Tetra(4-sulfonatophenyl) porphyrin chloride, to be encapsulated inside the DNA-tetrahedron. The porphyrins were successfully assembled inside the tetrahedron through coordination of two terminal histidines from the orthogonally oriented peptides covalently attached to the DNA. The assembly has been characterized using Electron Paramagnetic Resonance (EPR), optical spectroscopy, Dynamic Light Scattering (DLS), and x-ray crystallography. The results reveal that the spin state of the metal, iron (III), switches during assembly from the high-spin state to low-spin state.
ContributorsRendek, Kimberly Nicole (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2012
Interactions driving the collapse of islet amyloid polypeptide: implications for amyloid aggregation
Description
Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37-residue intrinsically disordered hormone involved in glucose regulation and gastric emptying. The aggregation of hIAPP into amyloid fibrils is believed to play a causal role in type 2 diabetes. To date, not much is known about the monomeric state of hIAPP or how it undergoes an irreversible transformation from disordered peptide to insoluble aggregate. IAPP contains a highly conserved disulfide bond that restricts hIAPP(1-8) into a short ring-like structure: N_loop. Removal or chemical reduction of N_loop not only prevents cell response upon binding to the CGRP receptor, but also alters the mass per length distribution of hIAPP fibers and the kinetics of fibril formation. The mechanism by which N_loop affects hIAPP aggregation is not yet understood, but is important for rationalizing kinetics and developing potential inhibitors. By measuring end-to-end contact formation rates, Vaiana et al. showed that N_loop induces collapsed states in IAPP monomers, implying attractive interactions between N_loop and other regions of the disordered polypeptide chain . We show that in addition to being involved in intra-protein interactions, the N_loop is involved in inter-protein interactions, which lead to the formation of extremely long and stable β-turn fibers. These non-amyloid fibers are present in the 10 μM concentration range, under the same solution conditions in which hIAPP forms amyloid fibers. We discuss the effect of peptide cyclization on both intra- and inter-protein interactions, and its possible implications for aggregation. Our findings indicate a potential role of N_loop-N_loop interactions in hIAPP aggregation, which has not previously been explored. Though our findings suggest that N_loop plays an important role in the pathway of amyloid formation, other naturally occurring IAPP variants that contain this structural feature are incapable of forming amyloids. For example, hIAPP readily forms amyloid fibrils in vitro, whereas the rat variant (rIAPP), differing by six amino acids, does not. In addition to being highly soluble, rIAPP is an effective inhibitor of hIAPP fibril formation . Both of these properties have been attributed to rIAPP's three proline residues: A25P, S28P and S29P. Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect conformational ensembles of intrinsically disordered proteins. The specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored, as the detection of such properties is experimentally challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High-resolution techniques able to measure tertiary contact formations are needed to address this issue. We employ a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. We explore the proline substitutions in IAPP and quantify their effects in terms of intrinsic chain stiffness. We find that the three proline mutations found in rIAPP increase chain stiffness. Interestingly, we also find that residue R18 plays an important role in rIAPP's unique chain stiffness and, together with the proline residues, is a determinant for its non-amyloidogenic properties. We discuss the implications of our findings on the role of prolines in IDPs.
ContributorsCope, Stephanie M (Author) / Vaiana, Sara M (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Ros, Robert (Committee member) / Lindsay, Stuart M (Committee member) / Ozkan, Sefika B (Committee member) / Arizona State University (Publisher)
Created2013
Description
Photosynthesis is the primary source of energy for most living organisms. Light harvesting complexes (LHC) play a vital role in harvesting sunlight and passing it on to the protein complexes of the electron transfer chain which create the electrochemical potential across the membrane which drives ATP synthesis. phycobilisomes (PBS) are the most important LHCs in cyanobacteria. PBS is a complex of three light harvesting proteins: phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). This work has been done on a newly discovered cyanobacterium called Leptolyngbya Heron Island (L.HI). This study has three important goals: 1) Sequencing, assembly and annotation of the L.HI genome - Since this is a newly discovered cyanobacterium, its genome was not previously elucidated. Illumina sequencing, a type of next generation sequencing (NGS) technology was employed to sequence the genome. Unfortunately, the natural isolate contained other contaminating and potentially symbiotic bacterial populations. A novel bioinformatics strategy for separating DNA from contaminating bacterial populations from that of L.HI was devised which involves a combination of tetranucleotide frequency, %(G+C), BLAST analysis and gene annotation. 2) Structural elucidation of phycoerythrin - Phycoerythrin is the most important protein in the PBS assembly because it is one of the few light harvesting proteins which absorbs green light. The protein was crystallized and its structure solved to a resolution of 2Å. This protein contains two chemically distinct types of chromophores: phycourobilin and phycoerythrobilin. Energy transfer calculations indicate that there is unidirectional flow of energy from phycourobilin to phycoerythrobilin. Energy transfer time constants using Forster energy transfer theory have been found to be consistent with experimental data available in literature. 3) Effect of chromatic acclimation on photosystems - Chromatic acclimation is a phenomenon in which an organism modulates the ratio of PE/PC with change in light conditions. Our investigation in case of L.HI has revealed that the PE is expressed more in green light than PC in red light. This leads to unequal harvesting of light in these two states. Therefore, photosystem II expression is increased in red-light acclimatized cells coupled with an increase in number of PBS.
ContributorsPaul, Robin (Author) / Fromme, Petra (Thesis advisor) / Ros, Alexandra (Committee member) / Roberson, Robert (Committee member) / Arizona State University (Publisher)
Created2014
Description
Membrane proteins are a vital part of cellular structure. They are directly involved in many important cellular functions, such as uptake, signaling, respiration, and photosynthesis, among others. Despite their importance, however, less than 500 unique membrane protein structures have been determined to date. This is due to several difficulties with macromolecular crystallography, primarily the difficulty of growing large, well-ordered protein crystals. Since the first proof of concept for femtosecond nanocrystallography showing that diffraction patterns can be collected on extremely small crystals, thus negating the need to grow larger crystals, there have been many exciting advancements in the field. The technique has been proven to show high spatial resolution, thus making it a viable method for structural biology. However, due to the ultrafast nature of the technique, which allows for a lack of radiation damage in imaging, even more interesting experiments are possible, and the first temporal and spatial images of an undamaged structure could be acquired. This concept was denoted as time-resolved femtosecond nanocrystallography.
This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process.
This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process.
ContributorsKupitz, Christopher (Author) / Fromme, Petra (Thesis advisor) / Spence, John C. (Thesis advisor) / Redding, Kevin (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2014
Description
Single molecule identification is one essential application area of nanotechnology. The application areas including DNA sequencing, peptide sequencing, early disease detection and other industrial applications such as quantitative and quantitative analysis of impurities, etc. The recognition tunneling technique we have developed shows that after functionalization of the probe and substrate of a conventional Scanning Tunneling Microscope with recognition molecules ("tethered molecule-pair" configuration), analyte molecules trapped in the gap that is formed by probe and substrate will bond with the reagent molecules. The stochastic bond formation/breakage fluctuations give insight into the nature of the intermolecular bonding at a single molecule-pair level. The distinct time domain and frequency domain features of tunneling signals were extracted from raw signals of analytes such as amino acids and their enantiomers. The Support Vector Machine (a machine-learning method) was used to do classification and predication based on the signal features generated by analytes, giving over 90% accuracy of separation of up to seven analytes. This opens up a new interface between chemistry and electronics with immediate implications for rapid Peptide/DNA sequencing and molecule identification at single molecule level.
ContributorsZhao, Yanan, 1986- (Author) / Lindsay, Stuart (Thesis advisor) / Nemanich, Robert (Committee member) / Qing, Quan (Committee member) / Ros, Robert (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2014
Description
Proteins and peptides fold into dynamic structures that access a broad functional landscape, however, designing artificial polypeptide systems continues to be a great chal-lenge. Conversely, deoxyribonucleic acid (DNA) engineering is now routinely used to build a wide variety of two dimensional and three dimensional (3D) nanostructures from simple hybridization based rules, and their functional diversity can be significantly ex-panded through site specific incorporation of the appropriate guest molecules. This dis-sertation describes a gentle methodology for using short (8 nucleotide) peptide nucleic acid (PNA) linkers to assemble polypeptides within a 3D DNA nanocage, as a proof of concept for constructing artificial catalytic centers. PNA-polypeptide conjugates were synthesized directly using microwave assisted solid phase synthesis or alternatively PNA linkers were conjugated to biologically expressed proteins using chemical crosslinking. The PNA-polypeptides hybridized to the preassembled DNA nanocage at room tempera-ture or 11 ⁰C and could be assembled in a stepwise fashion. Time resolved fluorescence anisotropy and gel electrophoresis were used to determine that a negatively charged az-urin protein was repelled outside of the negatively charged DNA nanocage, while a posi-tively charged cytochrome c protein was retained inside. Spectroelectrochemistry and an in-gel luminol oxidation assay demonstrated the cytochrome c protein remained active within the DNA nanocage and its redox potential decreased modestly by 10 mV due to the presence of the DNA nanocage. These results demonstrate the benign PNA assembly conditions are ideal for preserving polypeptide structure and function, and will facilitate the polypeptide-based assembly of artificial catalytic centers inside a stable DNA nanocage. A prospective application of assembling multiple cyclic γ-PNA-peptides to mimic the oxygen-evolving complex (OEC) catalytic active site from photosystem II (PSII) is described. In this way, the robust catalytic capacity of PSII could be utilized, without suffering the light-induced damage that occurs by the photoreactions within PSII via triplet state formation, which limits the efficiency of natural photosynthesis. There-fore, this strategy has the potential to revolutionize the process of designing and building robust catalysts by leveraging nature's recipes, and also providing a flexible and con-trolled artificial environment that might even improve them further towards commercial viability.
ContributorsFlory, Justin David (Author) / Fromme, Petra (Thesis advisor) / Yan, Hao (Committee member) / Buttry, Daniel (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Description
Biophysical techniques have been increasingly applied toward answering biological questions with more precision. Here, three different biological systems were studied with the goal of understanding their dynamic differences, either conformational dynamics within the system or oligomerization dynamics between monomers. With Cy3 on the 5' end of DNA, the effects of changing the terminal base pair were explored using temperature-dependent quantum yields. It was discovered, in combination with simulations, that a terminal thymine base has the weakest stacking interactions with the Cy3 dye compared to the other three bases. With ME1 heterodimers, the goal was to see if engineering a salt bridge at the dimerization interface could allow for control over dimerization in a pH-dependent manner. This was performed experimentally by measuring FRET between monomers containing either a Dap or an Asp mutation and comparing FRET efficiency at different pHs. It was demonstrated that the heterodimeric salt bridge would only form in a pH range near neutrality. Finally, with DNA processivity clamps, one aim was to compare the equilibrium dissociation constants, kinetic rate constants, and lifetimes of the closed rings for beta clamp and PCNA. This was done using a variety of biophysical techniques but with three as the main focus: fluorescence correlation spectroscopy, single-molecule experiments, and time-correlated single photon counting measurements. The stability of beta clamp was found to be three orders of magnitude higher when measuring solution stability but only one order of magnitude higher when measuring intrinsic stability, which is a result of salt bridge interactions in the interface of beta clamp. Ongoing work built upon the findings from this project by attempting to disrupt interface stability of different beta clamp mutants by adding salt or changing the pH of the solution. Lingering questions about the dynamics of different areas of the clamps has led to another project for which we have developed a control to demystify some unexpected similarities between beta clamp mutants. With that project, we show that single-labeled and double-labeled samples have similar autocorrelation decays in florescence correlation spectroscopy, allowing us to rule out the dyes themselves as causing fluctuations in the 10-100 microsecond timescale.
ContributorsBinder, Jennifer (Author) / Levitus, Marcia (Thesis advisor) / Wachter, Rebekka (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2015
Description
Calcitonin Gene-Related Peptide (CGRP) is an intrinsically disordered protein
that has no regular secondary structure, but plays an important role in vasodilation and pain transmission in migraine. Little is known about the structure and dynamics of the monomeric state of CGRP or how CGRP is able to function in the cell, despite the lack of regular secondary structure. This work focuses characterizing the non-local structural and dynamical properties of the CGRP monomer in solution, and understanding how these are affected by the sequence and the solution environment. The unbound, free state of CGRP is measured using a nanosecond laser-pump spectrophotometer, which allows measuring the end-to-end distance (a non-local structural property) and the rate of end-to-end contact formation (intra-chain diffusional dynamics). The data presented in this work show that electrostatic interactions strongly modulate the structure of CGRP, and that peptide-solvent interactions are sequence and charge dependent and can have a significant effect on the internal dynamics of the peptide. In the last few years migraine research has shifted focus to disrupting the CGRP-receptor pathway through the design of pharmacological drugs that bind to either CGRP or its receptor, inhibiting receptor activation and therefore preventing or reducing the frequency of migraine attacks. Understanding what types of intra- and inter-chain interactions dominate in CGRP can help better design drugs that disrupt the binding of CGRP to its receptor.
that has no regular secondary structure, but plays an important role in vasodilation and pain transmission in migraine. Little is known about the structure and dynamics of the monomeric state of CGRP or how CGRP is able to function in the cell, despite the lack of regular secondary structure. This work focuses characterizing the non-local structural and dynamical properties of the CGRP monomer in solution, and understanding how these are affected by the sequence and the solution environment. The unbound, free state of CGRP is measured using a nanosecond laser-pump spectrophotometer, which allows measuring the end-to-end distance (a non-local structural property) and the rate of end-to-end contact formation (intra-chain diffusional dynamics). The data presented in this work show that electrostatic interactions strongly modulate the structure of CGRP, and that peptide-solvent interactions are sequence and charge dependent and can have a significant effect on the internal dynamics of the peptide. In the last few years migraine research has shifted focus to disrupting the CGRP-receptor pathway through the design of pharmacological drugs that bind to either CGRP or its receptor, inhibiting receptor activation and therefore preventing or reducing the frequency of migraine attacks. Understanding what types of intra- and inter-chain interactions dominate in CGRP can help better design drugs that disrupt the binding of CGRP to its receptor.
ContributorsSizemore, Sara (Author) / Vaiana, Sara (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Ros, Robert (Committee member) / Lindsay, Stuart (Committee member) / Ozkan, Sefika (Committee member) / Arizona State University (Publisher)
Created2015
Description
Fluorescence spectroscopy is a popular technique that has been particularly useful in probing biological systems, especially with the invention of single molecule fluorescence. For example, Förster resonance energy transfer (FRET) is one tool that has been helpful in probing distances and conformational changes in biomolecules. In this work, important properties necessary in the quantification of FRET were investigated while FRET was also applied to gain insight into the dynamics of biological molecules. In particular, dynamics of damaged DNA was investigated. While damages in DNA are known to affect DNA structure, what remains unclear is how the presence of a lesion, or multiple lesions, affects the flexibility of DNA, especially in relation to damage recognition by repair enzymes. DNA conformational dynamics was probed by combining FRET and fluorescence anisotropy along with biochemical assays. The focus of this work was to investigate the relationship between dynamics and enzymatic repair. In addition, to properly quantify fluorescence and FRET data, photophysical phenomena of fluorophores, such as blinking, needs to be understood. The triplet formation of the single molecule dye TAMRA and the photoisomerization yield of two different modifications of the single molecule cyanine dye Cy3 were examined spectroscopically to aid in accurate data interpretation. The combination of the biophysical and physiochemical studies illustrates how fluorescence spectroscopy can be used to answer biological questions.
ContributorsShepherd Stennett, Elana Maria (Author) / Levitus, Marcia (Thesis advisor) / Ros, Robert (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2015
Description
Photosystem I (PSI) is a multi-subunit, pigment-protein complex that catalyzes light-driven electron transfer (ET) in its bi-branched reaction center (RC). Recently it was suggested that the initial charge separation (CS) event can take place independently within each ec2/ec3 chlorophyll pair. In order to improve our understanding of this phenomenon, we have generated new mutations in the PsaA and PsaB subunits near the electron transfer cofactor 2 (ec2 chlorophyll). PsaA-Asn604 accepts a hydrogen bond from the water molecule that is the axial ligand of ec2B and the case is similar for PsaB-Asn591 and ec2A. The second set of targeted sites was PsaA-Ala684 and PsaB-Ala664, whose methyl groups are present near ec2A and ec2B, respectively. We generated a number of mutants by targeting the selected protein residues. These mutations were expected to alter the energetics of the primary charge separation event.
The PsaA-A684N mutants exhibited increased ET on the B-branch as compared to the A-branch in both in vivo and in vitro conditions. The transient electron paramagnetic resonance (EPR) spectroscopy revealed the formation of increased B-side radical pair (RP) at ambient and cryogenic temperatures. The ultrafast transient absorption spectroscopy and fluorescence decay measurement of the PsaA-A684N and PsaB-A664N showed a slight deceleration of energy trapping. Thus making mutations near ec2 on each branch resulted into modulation of the charge separation process. In the second set of mutants, where ec2 cofactor was target by substitution of PsaA-Asn604 or PsaB-Asn591 to other amino acids, a drop in energy trapping was observed. The quantum yield of CS decreases in Asn to Leu and His mutants on the respective branch. The P700 triplet state was not observed at room and cryogenic temperature for these mutants, nor was a rapid decay of P700+ in the nanosecond timescale, indicating that the mutations do not cause a blockage of electron transfer from the ec3 Chl. Time-resolved fluorescence results showed a decrease in the lifetime of the energy trapping. We interpret this decrease in lifetime as a new channel of excitation energy decay, in which the untrapped energy dissipates as heat through a fast internal conversion process. Thus, a variety of spectroscopic measurements of PSI with point mutations near the ec2 cofactor further support that the ec2 cofactor is involved in energy trapping process.
The PsaA-A684N mutants exhibited increased ET on the B-branch as compared to the A-branch in both in vivo and in vitro conditions. The transient electron paramagnetic resonance (EPR) spectroscopy revealed the formation of increased B-side radical pair (RP) at ambient and cryogenic temperatures. The ultrafast transient absorption spectroscopy and fluorescence decay measurement of the PsaA-A684N and PsaB-A664N showed a slight deceleration of energy trapping. Thus making mutations near ec2 on each branch resulted into modulation of the charge separation process. In the second set of mutants, where ec2 cofactor was target by substitution of PsaA-Asn604 or PsaB-Asn591 to other amino acids, a drop in energy trapping was observed. The quantum yield of CS decreases in Asn to Leu and His mutants on the respective branch. The P700 triplet state was not observed at room and cryogenic temperature for these mutants, nor was a rapid decay of P700+ in the nanosecond timescale, indicating that the mutations do not cause a blockage of electron transfer from the ec3 Chl. Time-resolved fluorescence results showed a decrease in the lifetime of the energy trapping. We interpret this decrease in lifetime as a new channel of excitation energy decay, in which the untrapped energy dissipates as heat through a fast internal conversion process. Thus, a variety of spectroscopic measurements of PSI with point mutations near the ec2 cofactor further support that the ec2 cofactor is involved in energy trapping process.
ContributorsBadshah, Syed Lal (Author) / Redding, Kevin E (Thesis advisor) / Fromme, Petra (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2014