Matching Items (24)
Filtering by
- All Subjects: Biochemistry
- Creators: Wang, Xu
- Member of: Theses and Dissertations
Exploring Structure and Function of Human Cold Sensing Protein TRPM8 with ROSETTA Comparative Models
Description
Transient Receptor Potential (TRP) channels are a diverse class of ion channels notable as polymodal sensors. TRPM8 is a TRP channel implicated in cold sensation, nociception, and a variety of human diseases, including obesity and cancer. Despite sustained interest in TRPM8 since its discovery in 2001, many of the molecular mechanisms that underlie function are not yet clear. Knowledge of these properties could have implications for medicine and physiological understanding of sensation and signaling. Structures of TRP channels have proven challenging to solve, but recent Cryoelectron microscopy (Cryo-EM) structures of TRPV1 provide a basis for homology-based modeling of TRP channel structures and interactions. I present an ensemble of 11,000 Rosetta computational homology models of TRPM8 based on the recent Cryo-EM apo structure of TRPV1 (PDB code:3J5P). Site-directed mutagenesis has provided clues about which residues are most essential for modulatory ligands to bind, so the models presented provide a platform to investigate the structural basis of TRPM8 ligand modulation complementary to existing functional and structural information. Menthol and icilin appear to interact with interfacial residues in the sensor domain (S1-S4). One consensus feature of these sites is the presence of local contacts to the S4 helix, suggesting this helix may be mechanistically involved with the opening of the pore. Phosphatidylinositol 4,5-bisphosphate (PIP2)has long been known to interact with the C-terminus of TRPM8, and some of the homology models contain plausible binding pockets where PIP2 can come into contact with charged residues known to be essential for PIP2 modulation. Future in silico binding experiments could provide testable hypothesis for in vitro structural studies, and experimental data (e.g. distance constraints from electron paramagnetic resonance spectroscopy [EPR]) could further refine the models.
ContributorsHelsell, Cole Vincent Maher (Author) / Van Horn, Wade (Thesis director) / Wang, Xu (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Understanding glycosaminoglycans’ (GAG) interaction with proteins is of growing interest for therapeutic applications. For instance, heparin is a GAG exploited for its ability to inhibit proteases, therefore inducing anticoagulation. For this reason, heparin is extracted in mass quantities from porcine intestine in the pharmaceutical field. Following a contamination in 2008, alternative sources for heparin are desired. In response, much research has been invested in the extraction of the naturally occurring polysaccharide, heparosan, from Escherichia coli K5 strain. As heparosan contains the same structural backbone as heparin, modifications can be made to produce heparin or heparin-like molecules from this source. Furthermore, isotopically labeled batches of heparosan can be produced to aid in protein-GAG interaction studies. In this study, a comparative look between extraction and purification methods of heparosan was taken. Fed-batch fermentation of this E. coli strain followed by subsequent purification yielded a final 13C/15N labeled batch of 90mg/L of heparosan which was then N-sulfated. Furthermore, a labeled sulfated disaccharide from this batch was utilized in a protein interaction study with CCL5. With NMR analysis, it was found that this heparin-like molecule interacted with CCL5 when its glucosamine residue was in a β-conformation. This represents an interaction reliant on a specific anomericity of this GAG molecule.
ContributorsHoffman, Kristin Michelle (Author) / Wang, Xu (Thesis director) / Cabirac, Gary (Committee member) / Morgan, Ashli (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Integrin is a protein in cells that manage cell adhesion. They are crucial to the biochemical functions of cells. L 2 is one type of integrin. Its I domain is responsible for ligand binding. Scientists understand how Alpha L I domain binds Mg2+ at a pH of 7 but not in acidic environments. Knowing the specificity of integrin at a lower pH is important because when tissues become inflamed, they release acidic compounds. We have cloned, expressed, and purified L I-domain and using NMR analysis, we determined that wild type Alpha L I domain does not bind to Mg2+ at a pH of 5.
ContributorsALAM, RAHAT (Author) / Wang, Xu (Thesis director) / Podolnikova, Nataly (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Based on past studies, urinary glycan biomarkers have the potential to be used as diagnostic and prognostic markers for treatment purposes. This study brought into play the bottom-up glycan node analysis approach to analyze 39 urine samples from COVID-19 positive and negative individuals using gas chromatography-mass spectrometry (GC-MS) to determine potential urinary glycan biomarkers of COVID-19. Glycan node analysis involves chemically breaking down glycans in whole biospecimens in a way that conserves both monosaccharide identity and linkage information that facilitates the capture of unique glycan features as single analytical signals. Following data acquisition, the student t-test was done on all the nodes, but only four prominent nodes (t-Deoxyhexopyranose, 2,3-Gal, t-GlcNAc, and 3,6-GalNAc with respective p-values 0.03027, 0.03973, 0.0224, and 0.0004) were below the threshold p-value of 0.05 and showed some differences in the mean between both groups. To eliminate the probability of having false positive p-values, Bonferroni correction was done on the four nodes but only the 3,6-GalNAc node emerged as the only node that was below the newly adjusted p-value. Because sample analyses were done in batches, the Kruskal Wallis test was done to know if the batch effect was responsible for the observed lower relative concentration of 3,6-GalNAc in COVID-19 positive patients than in negative patients. A receiver operating characteristic curve (ROC) was plotted for the 3,6-GalNAc node and the area under the curve (AUC) was calculated to be 0.84, casting the 3,6-GalNAc node was a potential biomarker of COVID-19. 3,6-GalNAc largely arises from branched O-glycan core structures, which are abundant in mucin glycoproteins that line the urogenital tract. Lowered relative concentrations of 3,6-GalNAc in the urine of COVID-19 positive patients may be explained by compromised kidney function that allows non-mucinous glycoproteins from the blood to contribute a greater proportion of the relative glycan node signals than in COVID-19 negative patients. Future prospective clinical studies will be needed to validate both the biomarker findings and this hypothesis.
ContributorsEyonghebi Tanyi, Agbor (Author) / Borges, Chad R (Thesis advisor) / Mills, Jeremy H (Committee member) / Guo, Jia (Committee member) / Arizona State University (Publisher)
Created2023
Description
Exposure of liquid biospecimens like plasma and serum (P/S) to improper handling and storage can impact the integrity of biomolecules, potentially leading to apparent quantitative changes of important clinical proteins. An accurate and quick estimate of the quality of biospecimens employed in biomarker discovery and validation studies is essential to facilitating accurate conclusions. ΔS-Cys-Albumin is a marker of blood P/S exposure to thawed conditions that can quantitatively track the exposure of P/S to temperatures greater than their freezing point of -30 C. Reported here are studies carried out to evaluate the potential of ΔS-Cys-Albumin to track the stability of clinically important analytes present in P/S upon their exposure to thawed conditions. P/S samples obtained from both cancer-free donors and cancer patients were exposed to 23 C (room temperature), 4 C and -20 C degrees, and the degree to which the apparent concentrations of clinically relevant biomolecules present in P/S were impacted during the time it took ΔS-Cys-Albumin to reach zero was measured. Analyte concentrations measured by molecular interaction-based assays were significantly impacted when samples were exposed to the point where average ΔS-Cys-Albumin fell below 12% at each temperature. Furthermore, the percentage of proteins that became unstable with time under thawed conditions exhibited a strong inverse linear relationship to ΔS-Cys-Albumin, indicating that ΔS-Cys-Albumin can serve as an effective surrogate marker to track the stability of other clinically relevant proteins in plasma as well as to estimate the fraction of proteins that have been destabilized by exposure to thawed conditions, regardless of what the exposure temperature(s) may have been. These results indicated that P/S exposure to thawed conditions disrupts epitopes required for clinical protein quantification via molecular interaction-based assays. In continuation of this theme, a spurious binding event between two clinically important proteins, Apolipoprotein E (ApoE) and Interferon- (IFN) present in human plasma under in vitro experimental conditions is also reported. The interaction was confirmed to be evident only when ApoE was expressed in vitro with a Glutathione-S-Transferase (GST) fusion tag. Future steps required to find the exact manner in which the GST fusion tag facilitated the association between ApoE and IFNγ are discussed with emphasis on the possible pitfalls associated with using fusion proteins for studying novel protein-protein interactions.
ContributorsKapuruge, Erandi Prasadini (Author) / Borges, Chad R (Thesis advisor) / LaBaer, Joshua (Committee member) / Van Horn, Wade (Committee member) / Arizona State University (Publisher)
Created2021
Description
Osteocalcin (Oc) is the most abundant non-collagen protein found in the bone, but its precise function is still not completely understood. Three glutamic acid (Glu) residues within its sequence are sites for vitamin K-dependent post-translational modification, replacing a hydrogen with a carboxylate located at the γ-carbon position, converting these to γ-carboxyglutamic acid (Gla) residues. This modification confers increased binding of Oc to Ca2+ and hydroxyapatite matrix. Presented here, novel metal binding partners Mn2+, Fe3+, and Cr3+ of human Oc were determined, while the previously identified binders to (generally) non-human Oc, Ca2+, Mg2+, Pb2+ and Al3+ were validated as binders to human Oc by direct infusion mass spectrometry with all metals binding with higher affinity to the post-translationally modified form (Gla-Oc) compared to the unmodified form (Glu-Oc). Oc was also found to form pentamer (Gla-Oc) and pentamer and tetramer (Glu-Oc) homomeric self-assemblies in the absence of NaCl, which disassembled to monomers in the presence of near physiological Na+ concentrations. Additionally, Oc was found to form filamentous structures in vitro by negative stain TEM in the presence of increased Ca2+ titrations in a Gla- and pH-dependent manner. Finally, by combining circular dichroism spectroscopy to determine the fraction of Gla-Oc bound, and inductively-coupled plasma mass spectrometry to quantify total Al concentrations, the data were fit to a single-site binding model and the equilibrium dissociation constant for Al3+ binding to human Gla-Oc was determined (Kd = 1.0 ± 0.12 nM). Including citrate, a known competitive binder of Al3+, maintained Al in solution and enabled calculation of free Al3+ concentrations using a Matlab script to solve the complex set of linear equations. To further improve Al solubility limits, the pH of the system was lowered to 4.5, the pH during bone resorption. Complementary binding experiments with Glu-Oc were not possible due to the observed precipitation of Glu-Oc at pH 4.5, although qualitatively if Glu-Oc binds Al3+, it is with much lower affinity compared to Gla-Oc. Taken together, the results presented here further support the importance of post-translational modification, and thus adequate nutritional intake of vitamin K, on the binding and self-assembly properties of human Oc.
ContributorsThibert, Stephanie (Author) / Borges, Chad R (Thesis advisor) / LaBaer, Joshua (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Description
G protein coupled receptors (GPCRs) mediate various of physiologicalactivities which makes them significant drug targets. Determination of atomic level
structure of GPCRs facilitates the structure-based drug design. The most widely
used method currently for solving GPCR structure is still protein crystallography
especially lipidic cubic phase (LCP) crystallization. LCP could mimic the native
environment of membrane protein which stable the membrane proteins. Traditional
synchrotron source requires large size large size protein crystals (>30 micron) due to
the radiation damage during data collection. However, acquiring large sized protein
crystals is challenging and not guaranteed practically. In this study, a novel method
was developed which combined LCP technology and micro-electron diffraction
(MicroED) technology. LCP-MicroED technology was able to collect complete
diffraction data sets from serval submicron protein crystals and deliver high
resolution protein structures. This technology was first confirmed with soluble
protein crystals, proteinase K and small molecule crystals, cholesterol. Furthermore,
this novel method was applied to a human GPCR target, Î22- adrenergic receptor
(Î22AR). The structure model was successfully built which proved the feasibility of
applying LCP-MicroED method to GPCRs and other membrane proteins. Besides, in
this research, a novel human GPCR target, human histamine 4 receptor(H4R) was
studied. Different constructs were expressed, purified, and characterized. Some key
residuals that affect ligand binding were confirmed.
ContributorsJing, Liang (Author) / Mazor, Yuval (Thesis advisor) / Mills, Jeremy (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2022
Description
The integrin Mac-1 (αMβ2, CD11b/CD18) is an important adhesion receptorexpressed on macrophages and neutrophils. It plays a crucial role in phagocytosis, cell-cell
fusion, and cell migration. αMβ2 is also the most promiscuous integrin with over 100 known
ligands that span a broad range of physical and chemical attributes, many of which bind to
the inserted (I) domain from the αM subunit. The interaction of αMI-domain with cytokine
pleiotrophin (PTN) were determine. PTN is a cationic protein known to induce Mac-1-
mediated adhesion and migration in cells. The data showed that PTN’s interaction with
αMI-domain contains both divalent cation-dependent and independent mechanisms. In
particular, PTN’s N-terminal domain has weak interactions with the N/C-termini side of
αMI-domain using a metal-independent mechanism. However, stronger interaction is
achieved through the chelation of the divalent cation in the metal ion-dependent adhesion
site of active αMI-domain by PTN’s acidic residues. Although many acidic residues in PTN
can act as the chelator, active αMI-domain’s interaction with PTN’s E98 plays an especially
important role. NOE, chemical shift perturbation (CSP) data, and mutagenesis studies
showed residues near E98 are at the binding interface and the E98 mutation greatly reduced
binding affinity between two proteins. Interestingly, the CSP and MD simulation data
showed the binding interface can be supported by the interaction of PTN’s H95 with the
acidic clusters D242, E244, and D273 from αMI-domain, while PTN’s E66 form
electrostatic interaction with R208 and K245 from αMI-domain. The determined
recognition motif of αMI-domain for its ligands is (H/R/K)xxE. The ability to accommodate
the longer distance between E and (H, R, K) compared to the zwitterionic motif RGDii
explained how αMβ2 can interact with a large repertoire of ligands and be versatile in its
functional portfolio.
ContributorsNguyen, Hoa Thi Thanh (Author) / Wang, Xu (Thesis advisor) / Van Horn, Wade (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2024
Description
Exposure of blood plasma/serum (P/S) to thawed conditions, greater than -30°C, can produce biomolecular changes that misleadingly impact measurements of clinical markers within archived samples. Reported here is a low sample-volume, dilute-and-shoot, intact protein mass spectrometric assay of albumin proteoforms called “ΔS-Cys-Albumin” that quantifies cumulative exposure of archived P/S samples to thawed conditions. The assay uses the fact that S-cysteinylation (oxidation) of albumin in P/S increases to a maximum value when exposed to temperatures greater than -30°C. The multi-reaction rate law that governs this albumin S-cysteinylation formation in P/S was determined and was shown to predict the rate of formation of S-cysteinylated albumin in P/S samples—a step that enables back-calculation of the time at which unknown P/S specimens have been exposed to room temperature. To emphasize the capability of this assay, a blind challenge demonstrated the ability of ΔS-Cys-Albumin to detect exposure of individual and grouped P/S samples to unfavorable storage conditions. The assay was also capable of detecting an anomaly in a case study of nominally pristine serum samples collected under NIH-sponsorship, demonstrating that empirical evidence is required to guarantee accurate knowledge of archived P/S biospecimen storage history.
The ex vivo glycation of human serum albumin was also investigated showing that P/S samples stored above their freezing point leads to significant increases in glycated albumin. These increases were found to occur within hours at room temperature, and within days at -20 °C. These increases continued over a period of 1-2 weeks at room temperature and over 200 days at -20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin—suggesting a role for oxidative glycation in the ex vivo glycation of albumin.
The ex vivo glycation of human serum albumin was also investigated showing that P/S samples stored above their freezing point leads to significant increases in glycated albumin. These increases were found to occur within hours at room temperature, and within days at -20 °C. These increases continued over a period of 1-2 weeks at room temperature and over 200 days at -20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin—suggesting a role for oxidative glycation in the ex vivo glycation of albumin.
ContributorsJeffs, Joshua W (Author) / Borges, Chad R (Thesis advisor) / Van Horn, Wade (Committee member) / Williams, Peter (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