Matching Items (7)
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
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
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 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.
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
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 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.
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
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
Description
Transient receptor potential vanilloid member 1 (TRPV1) is a membrane protein ion channel that functions as a heat and capsaicin receptor. In addition to activation by hot temperature and vanilloid compounds such as capsaicin, TRPV1 is modulated by various stimuli including acidic pH, endogenous lipids, diverse biological and synthetic chemical ligands, and modulatory proteins. Due to its sensitivity to noxious stimuli such as high temperature and pungent chemicals, there has been significant evidence that TRPV1 participates in a variety of human physiological and pathophysiological pathways, raising the potential of TRPV1 as an attractive therapeutic target. However, the polymodal nature of TRPV1 function has complicated clinical application because the TRPV1 activation mechanisms from different modes have generally been enigmatic. Consequently, tremendous efforts have put into dissecting the mechanisms of different activation modes, but numerous questions remain to be answered.
The studies conducted in this dissertation probed the role of the S1-S4 membrane domain in temperature and ligand activation of human TRPV1. Temperature-dependent solution nuclear magnetic resonance (NMR) spectroscopy for thermodynamic and mechanistic studies of the S1-S4 domain. From these results, a potential temperature sensing mechanism of TRPV1, initiated from the S1-S4 domain, was proposed. Additionally, direct binding of various ligands to the S1-S4 domain were used to ascertain the interaction site and the affinities (Kd) of various ligands to this domain. These results are the first to study the isolated S1-S4 domain of human TRPV1 and many results indicate that the S1-S4 domain is crucial for both temperature-sensing and is the general receptor binding site central to chemical activation.
The studies conducted in this dissertation probed the role of the S1-S4 membrane domain in temperature and ligand activation of human TRPV1. Temperature-dependent solution nuclear magnetic resonance (NMR) spectroscopy for thermodynamic and mechanistic studies of the S1-S4 domain. From these results, a potential temperature sensing mechanism of TRPV1, initiated from the S1-S4 domain, was proposed. Additionally, direct binding of various ligands to the S1-S4 domain were used to ascertain the interaction site and the affinities (Kd) of various ligands to this domain. These results are the first to study the isolated S1-S4 domain of human TRPV1 and many results indicate that the S1-S4 domain is crucial for both temperature-sensing and is the general receptor binding site central to chemical activation.
ContributorsKim, Minjoo (Author) / Van Horn, Wade D (Thesis advisor) / Wang, Xu (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2019
Description
Enzymes keep life nicely humming along by catalyzing important reactions at relevant timescales. Despite their immediate importance, how enzymes recognize and bind their substrate in a sea of cytosolic small molecules, carry out the reaction, and release their product in microseconds is still relatively opaque. Methods to elucidate enzyme substrate specificity indicate that the shape of the active site and the amino acid residues therein play a major role. However, lessons from Directed Evolution experiments reveal the importance of residues far from the active site in modulating substrate specificity. Enzymes are dynamic macromolecules composed of networks of interactions integrating the active site, where the chemistry occurs, to the rest of the protein. The objective of this work is to develop computational methods to modify enzyme ligand specificity, either through molding the active site to accommodate a novel ligand, or by identifying distal mutations that can allosterically alter specificity. To this end, two homologues in the β-lactamase family of enzymes, TEM-1, and an ancestrally reconstructed variant, GNCA, were studied to identify whether the modulation of position-specific distal-residue flexibility could modify ligand specificity. RosettaDesign was used to create TEM-1 variants with altered dynamic patterns. Experimental characterization of ten designed proteins indicated that mutations to residues surrounding rigid, highly coupled residues substantially affected both enzymatic activity and stability. In contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Five of the TEM-1 variants were crystallized to see if the changes in function observed were due to architectural changes to the active site. In a second project, a computational platform using RosettaDesign was developed to remodel the firefly luciferase active site to accommodate novel luciferins. This platform resulted in the development of five luciferin-luciferase pairs with red-shifted emission maxima, ready for multicomponent bioluminescent imaging applications in tissues. Although the projects from this work focus on two classes of proteins, they provide insight into the structure-function relationship of ligand specificity in enzymes and are broadly applicable to other systems.
ContributorsKolbaba Kartchner, Bethany (Author) / Mills, Jeremy H (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Van Horn, Wade D (Committee member) / Arizona State University (Publisher)
Created2023