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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
Description
The evolution of photosynthesis caused the oxygen-rich atmosphere in which we thrive today. Although the reaction centers involved in oxygenic photosynthesis probably evolved from a protein like the reaction centers in modern anoxygenic photosynthesis, modern anoxygenic reaction centers are poorly understood. One such anaerobic reaction center is found in Heliobacterium modesticaldum. Here, the photosynthetic properties of H. modesticaldum are investigated, especially as they pertain to its unique photochemical reaction center.
The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.
In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.
The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.
In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.
ContributorsGisriel, Christopher J (Author) / Redding, Kevin E (Thesis advisor) / Jones, Anne K (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2017
Description
Rubisco activase (Rca) from higher plants is a stromal ATPase essential for reactivating Rubiscos rendered catalytically inactive by endogenous inhibitors. Rca’s functional state is thought to consist of ring-like hexameric assemblies, similar to other members of the AAA+ protein superfamily. However, unlike other members, it does not form obligate hexamers and is quite polydisperse in solution, making elucidation of its self-association pathway challenging. This polydispersity also makes interpretation of traditional biochemical approaches difficult, prompting use of a fluorescence-based technique (Fluorescence Correlation Spectroscopy) to investigate the relationship between quaternary structure and function. Like cotton β Rca, tobacco β Rca appears to assemble in a step-wise and nucleotide-dependent manner. Incubation in varying nucleotides appears to alter the equilibrium between varying oligomers, either promoting or minimizing the formation of larger oligomers. High concentrations of ADP seem to favor continuous assembly towards larger oligomers, while assembly in the presence of ATP-yS (an ATP analog) appears to halt continuous assembly in favor of hexameric species. In contrast, assembly in the “Active ATP Turnover” condition (a mixture of ATP and ADP) appears to favor an almost equal distribution of tetramer and hexamer, which when compared with ATPase activity, shows great alignment with maximum activity in the low µM range. Despite this alignment, the decrease in ATPase activity does not follow any particular oligomer, but rather decreases with increasing aggregation, suggesting that assembly dynamics may regulate ATPase activity, rather than the formation/disappearance of one specific oligomer. Work presented here also indicates that all oligomers larger than hexamers are catalytically inactive, thus providing support for the idea that they may serve as a storage mechanism to minimize wasteful hydrolysis. These findings are also supported by assembly work carried out on an Assembly Mutant (R294V), known for favoring formation of closed-ring hexamers. Similar assembly studies were carried out on spinach Rca, however, due to its aggregation propensity, FCS results were more difficult to interpret. Based on these findings, one could argue that assembly dynamics are essential for Rca function, both in ATPase and in regulation of Rubisco carboxylation activity, thus providing a rational for Rca’s high degree of polydispersity.
ContributorsSerban, Andrew J (Author) / Wachter, Rebekka M. (Thesis advisor) / Levitus, Marcia (Thesis advisor) / Redding, Kevin E (Committee member) / Van Horn, Wade D (Committee member) / Arizona State University (Publisher)
Created2018
Description
Due to the COVID-19 pandemic, declared in March of 2020, there have been many lifestyle changes which have likely influenced tobacco smoking behavior. Such lifestyle changes include lockdowns, stay at home orders, reduction in social cues related to smoking, increased stress, and boredom among other things. This study utilized a cross-sectional survey which looked into these behaviors, primarily perceived risk to COVID-19, and determined if there is an association between perceived risk and education level/race. Education level is a proxy for income and material resources, therefore making it more likely that people with lower levels of education have fewer resources and higher perceived risk to negative effects of COVID-19. Additionally, people of color are often marginalized in the medical community along with being the target of heavy advertising by tobacco companies which have likely impacted risk to COVID-19 as well.
ContributorsLodha, Pratishtha (Author) / Leischow, J. Scott (Thesis director) / Pearson, Jennifer (Committee member) / School of Life Sciences (Contributor) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Receiving signals and responding to the environment is crucial for survival for every living organism. One of those signals is being able to detect environmental and visceral temperatures. Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential melastatin 8 (TRPM8) are ion channels within cells that allow higher organisms to detect hot and cold temperatures, respectively. These TRP channels are also implicated in diverse physiological roles including pain, obesity, and cancer. As a result, these channels have garnered interest as potential targets for therapeutic interventions. However, the entanglement of TRPV1 and TRPM8 polymodal activation where it responds to a variety of different stimuli has caused adverse side effects of body thermal dysregulation and misregulation when antagonizing these channels as drug targets. This dissertation will dissect the molecular mechanism and regulation of TRPV1 and TRPM8. An in-depth look into the complex and conflicting results in trying to find the key area for thermosensation as well as looking into disentangling the polymodal activation modes in TRPV1. The regulatory mechanism between TRPM8 with phosphoinositide interacting regulator of TRPs (PIRT) and calmodulin will be examined using nuclear magnetic resonance (NMR). A computational, experimental, and methodical approach into ancestral TRPM8 orthologs using whole-cell patch-clamp electrophysiology, calcium mobilization assay, and cellular thermal shift assay (CETSA) to determine whether these modes of activation can be decoupled. Lastly, smaller studies are covered like developing a way to delivery full-length and truncated protein using amphipols to artificial and live cells without the biological regulatory processes and the purification of the TRPM8 transmembrane domain (TMD). In the end, two successful methods were developed to study the polymodal activation of proteins.
ContributorsLuu, Dustin Dean (Author) / Van Horn, Wade D (Thesis advisor) / Redding, Kevin E (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2023