Matching Items (12)
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Description
The green fluorescent protein (GFP)-like fluorescent proteins play an important role for the color of reef-building corals. Different colors of extant coral fluorescent proteins (FPs) have evolved from a green ancestral protein. Interestingly, green-to-red photoconversion FPs (Kaede-type Red FPs) are only found in clade D from Scleractinia (Faviina suborder). Therefore,

The green fluorescent protein (GFP)-like fluorescent proteins play an important role for the color of reef-building corals. Different colors of extant coral fluorescent proteins (FPs) have evolved from a green ancestral protein. Interestingly, green-to-red photoconversion FPs (Kaede-type Red FPs) are only found in clade D from Scleractinia (Faviina suborder). Therefore, I focus on the evolution of Kaede-type FPs from Faviina suborder ancestral FP. A total of 13 mutations have been identified previously that recapitulate the evolution of Kaede-type red FPs from the ancestral green FP. To examine the effect of each mutation, total ten reconstructed FPs were analyzed and six x-ray crystal structures were solved. These substitutions created a more hydrophilic environment around the carbonyl group of Phe61. Also, they increased the flexibility of the c-terminal chain, which keeps it from interacting with the entrance of the putative solvent channel. The photoconversion reaction shows a twophase kinetics. After the rapid initial phase, the overall reaction followed the firstorder kinetics. Based on the crystal structure analysis, I propose a new mechanism for Kaede-type FP photoconversion process, which a proton transfers via Gln38 to the carbonyl group of Phe61.
ContributorsKim, Hanseong (Author) / Wachter, Rebekka M. (Thesis advisor) / Fromme, Petra (Committee member) / Redding, Kevin E (Committee member) / Arizona State University (Publisher)
Created2012
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Description
Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties

Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties of the proteins derived from GFP allows increased complexity of experiments and consequently, information content of the data acquired. The importance of arginine-96 in GFP has been widely discussed. It has been established as vital to the kinetics of chromophore maturation and to the overall fold of GFP before post-translational self-modification. Its value during chromophore maturation has been demonstrated by mutational studies and a hypothesis proposed for its catalytic function. A strategy is described herein to determine its pKa value via NMR to determine whether Arg96 possesses the chemical capacity to function as a general base during GFP chromophore biosynthesis. Förster resonance energy transfer (FRET) techniques commonly employ Enhanced Cyan Fluorescent Proteins (ECFPs) and their derivatives as donor fluorophores useful in real-time, live-cell imaging. These proteins have a tryptophan-derived chromophore that emits light in the blue region of the visible spectrum. Most ECFPs suffer from fluorescence instability, which, coupled with their low quantum yield, makes data analysis unreliable. The structural heterogeneity of these proteins also results in undesirable photophysical characteristics. Recently, mCerulean3, a ten amino acid mutant of ECFP, was introduced as an optimized FRET-donor protein (1). The amino acids changed include a mobile residue, Asp148, which has been mutated to a glycine in the new construct, and Thr65 near the chromophore has been mutated to a serine, the wild-type residue at this location. I have solved the x-ray crystal structure of mCerulean3 at low pH and find that the pH-dependent isomerization has been eliminated. The chromophore is in the trans-conformation previously observed in Cerulean at pH 8. The mutations that increase the quantum yield and improve fluorescence brightness result in a stable, bright donor fluorophore well-suited for use in quantitative microscopic imaging.
ContributorsWatkins, Jennifer L (Author) / Wachter, Rebekka M. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2012
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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

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
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Description
To mimic the membrane environment for the photosynthetic reaction center of the photoheterotrophic Heliobacterium modesticaldum, a proteoliposome system was developed using the lipids found in native membranes, as well as a lipid possessing a Ni(II)-NTA head group. The liposomes were also saturated with menaquinone-9 to provide further native conditions, given

To mimic the membrane environment for the photosynthetic reaction center of the photoheterotrophic Heliobacterium modesticaldum, a proteoliposome system was developed using the lipids found in native membranes, as well as a lipid possessing a Ni(II)-NTA head group. The liposomes were also saturated with menaquinone-9 to provide further native conditions, given that menaquinone is active within the heliobacterial reaction center in some way. Purified heliobacterial reaction center was reconstituted into the liposomes and a recombinant cytochrome c553 was decorated onto the liposome surface. The native lipid-attachment sequence of cytochrome c553 was truncated and replaced with a hexahistidine tag. Thus, the membrane-anchoring observed in vivo was simulated through the histidine tag of the recombinant cytochrome binding to the Ni(II)-NTA lipid's head group. The kinetics of electron transfer in this system was measured and compared to native membranes using transient absorption spectroscopy. The preferential-orientation of reconstituted heliobacterial reaction center was also measured by monitoring the proteoliposome system's ability to reduce a soluble acceptor, flavodoxin, in both whole and detergent-solubilized proteoliposome conditions. These data demonstrate that this proteoliposome system is reliable, biomimetic, and efficient for selectively testing the function of the photosynthetic reaction center of Heliobacterium modesticaldum and its interactions with both donors and acceptors. The recombinant cytochrome c553 performs similarly to native cytochrome c553 in heliobacterial membranes. These data also support the hypothesis that the orientation of the reconstituted reaction center is inherently selective for its bacteriochlorophyll special pair directed to the outer-leaflet of the liposome.
ContributorsJohnson, William Alexander (Author) / Redding, Kevin E (Thesis advisor) / Van Horn, Wade D (Committee member) / Jones, Anne K (Committee member) / Arizona State University (Publisher)
Created2018
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Description
The physiological phenomenon of sensing temperature is detected by transient

receptor (TRP) ion channels, which are pore forming proteins that reside in the

membrane bilayer. The cold and hot sensing TRP channels named TRPV1 and TRPM8

respectively, can be modulated by diverse stimuli and are finely tuned by proteins and

lipids. PIRT (phosphoinositide interacting

The physiological phenomenon of sensing temperature is detected by transient

receptor (TRP) ion channels, which are pore forming proteins that reside in the

membrane bilayer. The cold and hot sensing TRP channels named TRPV1 and TRPM8

respectively, can be modulated by diverse stimuli and are finely tuned by proteins and

lipids. PIRT (phosphoinositide interacting regulator of TRP channels) is a small

membrane protein that modifies TRPV1 responses to heat and TRPM8 responses to cold.

In this dissertation, the first direct measurements between PIRT and TRPM8 are

quantified with nuclear magnetic resonance and microscale thermophoresis. Using

Rosetta computational biology, TRPM8 is modeled with a regulatory, and functionally

essential, lipid named PIP2. Furthermore, a PIRT ligand screen identified several novel

small molecular binders for PIRT as well a protein named calmodulin. The ligand

screening results implicate PIRT in diverse physiological functions. Additionally, sparse

NMR data and state of the art Rosetta protocols were used to experimentally guide PIRT

structure predictions. Finally, the mechanism of thermosensing from the evolutionarily

conserved sensing domain of TRPV1 was investigated using NMR. The body of work

presented herein advances the understanding of thermosensing and TRP channel function

with TRP channel regulatory implications for PIRT.
ContributorsSisco, Nicholas John (Author) / Van Horn, Wade D (Thesis advisor) / Mills, Jeremy H (Committee member) / Wang, Xu (Committee member) / Yarger, Jeff L (Committee member) / Arizona State University (Publisher)
Created2018
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Description
All organisms need to be able to sense and respond to their environment. Much of this process takes place via proteins embedded in the cell membrane, the border between a living thing and the external world. Transient receptor potential (TRP) ion channels are a superfamily of membrane proteins that play

All organisms need to be able to sense and respond to their environment. Much of this process takes place via proteins embedded in the cell membrane, the border between a living thing and the external world. Transient receptor potential (TRP) ion channels are a superfamily of membrane proteins that play diverse roles in physiology. Among the 27 TRP channels found in humans and other animals, TRP melastatin 8 (TRPM8) and TRP vanilloid 1 (TRPV1) are the primary sensors of cold and hot temperatures, respectively. They underlie the molecular basis of somatic temperature sensation, but beyond this are also known to be involved in body temperature and weight regulation, inflammation, migraine, nociception, and some types of cancer. Because of their broad physiological roles, these channels are an attractive target for potential therapeutic interventions.

This dissertation presents experimental studies to elucidate the mechanisms underlying TRPM8 and TRPV1 function and regulation. Electrophysiology experiments show that modulation of TRPM8 activity by phosphoinositide interacting regulator of TRP (PIRT), a small membrane protein, is species dependent; human PIRT attenuates TRPM8 activity, whereas mouse PIRT potentiates the channel. Direct binding experiments and chimeric mouse-human TRPM8 channels reveal that this regulation takes place via the transmembrane domain of the channel. Ligand activation of TRPM8 is also investigated. A mutation in the linker between the S4 and S5 helices is found to generally decrease TRPM8 currents, and to specifically abrogate functional response to the potent agonist icilin without affecting icilin binding.

The heat activation thermodynamics of TRPV1 are also probed using temperature-controlled electrophysiology. The magnitude of the gating enthalpy of human TRPV1 is found to be similar to other species reported in the literature. Human TRPV1 also features an apparent heat inactivation process that results in reduced heat sensitivity after exposure to elevated temperatures. The work presented in this dissertation sheds light on the varied mechanisms of thermosensitive TRP channel function and regulation.
ContributorsHilton, Jacob Kenneth (Author) / Van Horn, Wade D (Thesis advisor) / Levitus, Marcia (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2019
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Description
ABSTRACT



The catalytic chaperone of Rubisco is AAA+ protein Rubisco activase (Rca), which hydrolyzes ATP and thus undergoes conformational change, helping in reactivating Rubisco. Rca reactivates Rubisco plausibly by removing its C- terminal tail from the opening of its active site thus releasing the inhibitor, a sugar phosphate molecule.

ABSTRACT



The catalytic chaperone of Rubisco is AAA+ protein Rubisco activase (Rca), which hydrolyzes ATP and thus undergoes conformational change, helping in reactivating Rubisco. Rca reactivates Rubisco plausibly by removing its C- terminal tail from the opening of its active site thus releasing the inhibitor, a sugar phosphate molecule. Rubisco and Rca are regulated by the stromal environment, which includes the ATP/ADP ratio, Mg2+ concentration, redox potential etc. Here the mechanistic regulation of tobacco β-Rca was studied using steady state enzyme kinetics in terms of product inhibition, Mg2+ activation, cooperativity and asymmetry. A continuous Pi measurement assay was developed, and using this assay catalytic parameters were obtained, such as kcat 20.6 ± 6.5 min-1 ( n = 9) and KM 0.113 ± 0.033 mM (n = 4). A Mg2+ induced increase of substrate affinity in Rca was observed, where the KM changes from 0.452 mM to 0.069 mM, with the changing of free Mg2+ concentration from 0.1 mM to 10 mM. Fitting the catalytic efficiency as a function free Mg2+ concentration by use of a binding model gave a Hill coefficient of 2.2, which indicates a secondary magnesium binding site on the enzyme. A 8.4 fold increase of catalytic efficiency with increasing magnesium from 0.1 mM to 6.5 mM suggests a significant Mg2+ induced regulation of Rca. Moderate product inhibition was observed in inhibition study (Ki = 0. 063 ± 0.018 mM). A positive cooperativity (nH = 2.1) in ATP hydrolysis between two subunits was observed in the presence of 0.132 mM ADP, but not in the absence of ADP. This indicated the presence of two different classes of subunits, suggesting an asymmetric model for the enzyme. Inhibited Rubisco (ER) up to 20 μM concentration did not affect ATPase activity, in line with previous reports. The concentration dependent correlation of Rca activity (tobacco β-Rca) and oligomerization (cotton β-Rca) suggested that the dimer maybe the most active oligomeric species. A nucleotide induced thermal stabilization of Rca was observed, where ADP is more stabilizing than ATP in the absence of Mg2+. Mg2+ has a small destabilizing effect alone and in presence of the ADP, but a stabilizing effect in presence of ATP. The ligand induced thermal stability was similar for cotton and tobacco β-Rca.
ContributorsHazra, Suratna (Author) / Wachter, Rebekka M. (Thesis advisor) / Fromme, Petra (Committee member) / Frasch, Wayne D (Committee member) / Arizona State University (Publisher)
Created2015
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Description
The primary carbon fixing enzyme Rubisco maintains its activity through release of trapped inhibitors by Rubisco activase (Rca). Very little is known about the interaction, but binding has been proposed to be weak and transient. Extensive effort was made to develop Förster resonance energy transfer (FRET) based assays to understand

The primary carbon fixing enzyme Rubisco maintains its activity through release of trapped inhibitors by Rubisco activase (Rca). Very little is known about the interaction, but binding has been proposed to be weak and transient. Extensive effort was made to develop Förster resonance energy transfer (FRET) based assays to understand the physical interaction between Rubisco and Rca, as well as understand subunit exchange in Rca.

Preparations of labeled Rubisco and Rca were utilized in a FRET-based binding assay. Although initial data looked promising, this approach was not fruitful, as no true FRET signal was observed. One possibility is that under the conditions tested, Rca is not able to undergo the structural reorganizations necessary to achieve binding-competent conformations. Rca may also be asymmetric, leading to less stable binding of an already weak interaction.

To better understand the structural adjustments of Rca, subunit exchange between different oligomeric species was examined. It was discovered that subunit exchange is nucleotide dependent, with ADP giving the fastest exchange, ATP giving slower exchange and ATPS inhibiting exchange. Manganese, like ADP, destabilizes subunit-subunit interactions for rapid and facile exchange between oligomers. Three different types of assemblies were deduced from the rates of subunit exchange: rigid types with extremely slow dissociation of individual protomers, tight assemblies with the physiological substrate ATP, and loose assemblies that provide fast exchange due to high ADP.

Information gained about Rca subunit exchange can be used to reexamine the physical interaction between Rubisco and Rca using the FRET-binding assay. These binding assays will provide insight into Rca states able to interact with Rubisco, as well as define conditions to generate bound states for structural analysis. In combination with assembly assays, subunit exchange assays and reactivation studies will provide critical information about the structure/function relationship of Rca in the presence of different nucleotides. Together, these FRET-based assays will help to characterize the Rca regulation mechanism and provide valuable insight into the Rubisco reactivation mechanism.
ContributorsForbrook, Dayna S (Author) / Wachter, Rebekka M. (Thesis advisor) / Allen, James (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2017
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Description
Telomerase is a special reverse transcriptase that extends the linear chromosome termini in eukaryotes. Telomerase is also a unique ribonucleoprotein complex which is composed of the protein component called Telomerase Reverse Transcriptase (TERT) and a telomerase RNA component (TR). The enzyme from most vertebrate species is able to utilize a

Telomerase is a special reverse transcriptase that extends the linear chromosome termini in eukaryotes. Telomerase is also a unique ribonucleoprotein complex which is composed of the protein component called Telomerase Reverse Transcriptase (TERT) and a telomerase RNA component (TR). The enzyme from most vertebrate species is able to utilize a short template sequence within TR to synthesize a long stretch of telomeric DNA, an ability termed "repeat addition processivity". By using human telomerase reconstituted both in vitro (Rabbit Reticulocyte Lysate) and in vivo (293FT cells), I have demonstrated that a conserved motif in the reverse transcriptase domain of the telomerase protein is crucial for telomerase repeat addition processivity and rate. Furthermore, I have designed a "template-free" telomerase to show that RNA/DNA duplex binding is a critical step for telomere repeat synthesis. In an attempt to expand the understanding of vertebrate telomerase, I have studied RNA-protein interactions of telomerase from teleost fish. The teleost fish telomerase RNA (TR) is by far the smallest vertebrate TR identified, providing a valuable model for structural research.
ContributorsXie, Mingyi (Author) / Chen, Julian J.L. (Thesis advisor) / Yan, Hao (Committee member) / Wachter, Rebekka M. (Committee member) / Arizona State University (Publisher)
Created2010
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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

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