Matching Items (49)
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- All Subjects: Biochemistry
- Creators: School of Molecular Sciences
- Creators: Van Horn, Wade
- Resource Type: Text
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
Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few TRP channels responsible for thermosensing. Despite sustained interest in the channel, the mechanisms underlying TRPM8 activation, modulation, and gating have proved challenging to study and remain poorly understood. In this thesis, I offer data collected on various expression, extraction, and purification conditions tested in E. Coli expression systems with the aim to optimize the generation of a structurally stable and functional human TRPM8 pore domain (S5 and S6) construct for application in structural biology studies. These studies, including the biophysical technique nuclear magnetic spectroscopy (NMR), among others, will be essential for elucidating the role of the TRPM8 pore domain in in regulating ligand binding, channel gating, ion selectively, and thermal sensitivity. Moreover, in the second half of this thesis, I discuss the ligation-independent megaprimer PCR of whole-plasmids (MEGAWHOP PCR) cloning technique, and how it was used to generate chimeras between TRPM8 and its nearest analog TRPM2. I review steps taken to optimize the efficiency of MEGAWHOP PCR and the implications and unique applications of this novel methodology for advancing recombinant DNA technology. I lastly present preliminary electrophysiological data on the chimeras, employed to isolate and study the functional contributions of each individual transmembrane helix (S1-S6) to TRPM8 menthol activation. These studies show the utility of the TRPM8\u2014TRPM2 chimeras for dissecting function of TRP channels. The average current traces analyzed thus far indicate that the S2 and S3 helices appear to play an important role in TRPM8 menthol modulation because the TRPM8[M2S2] and TRPM8[M2S3] chimeras significantly reduce channel conductance in the presence of menthol. The TRPM8[M2S4] chimera, oppositely, increases channel conductance, implying that the S4 helix in native TRPM8 may suppress menthol modulation. Overall, these findings show that there is promise in the techniques chosen to identify specific regions of TRPM8 crucial to menthol activation, though the methods chosen to study the TRPM8 pore independent from the whole channel may need to be reevaluated. Further experiments will be necessary to refine TRPM8 pore solubilization and purification before structural studies can proceed, and the electrophysiology traces observed for the chimeras will need to be further verified and evaluated for consistency and physiological significance.
ContributorsWaris, Maryam Siddika (Author) / Van Horn, Wade (Thesis director) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of English (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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
In this thesis, glycan nodes, the basic subunits of complex biological sugars, were studied to determine the reproducibility of gas chromatography-mass spectrometry (GC/MS) based methylation analysis of whole blood plasma by normalization using an internal standard of heavy permethylated glycans. Glycans are complex biological sugars that have a variety of applications in the human body and will display aberrant compositions when produced by cancerous cells. Thus an assay to determine their composition can be used as a diagnostic tool. It was shown that the assay may have potential use, but needs further refinement to become an improvement over current methods by analyzing the results of ratio-determination and replicate experiments.
ContributorsMiyasaki, Tyler Takeo (Author) / Borges, Chad (Thesis director) / Van Horn, Wade (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05
Description
The primary objective of this project is to further the knowledge about SCL26 family of anion transporters. The goals of the experiment were to find the lowest sulfate concentration where the yeast without Sulp1 and Sulp2 is able to grow, but it grows very slowly, and to find a higher sulfate concentration where the yeast grows quickly, with or without the sulfate transporters. The lowest sulfate concentration where the yeast without the sulfate transporters is able to grow was determined to be 2-4 mM, however, this range can likely be refined by more quantitative analytical methods. At a sulfate concentration of 20 mM sulfate or higher, the yeast is able to grow quickly without high-affinity sulfate transporters. The next step in the project is to re-introduce the Sulp1 and Sulp2 genes into the yeast, so that growth in low and high sulfate conditions can be compared with and without the Sulp1 and Sulp2 proteins. The long-term goals of the project are to bring experience with yeast to Dr. Nannenga’s structural discovery lab, to determine if yeast sulfate transporters respond in the same way to drug candidates as human sulfate transporters, and to determine the structure of the proteins using cryo-electron microscopy.
ContributorsCall, Nicolas I (Author) / Nannenga, Brent (Thesis director) / Wang, Xuan (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The devastating 2014 Ebola virus outbreak in Western Africa demonstrated the lack of therapeutic approaches available for the virus. Although monoclonal antibodies (mAb) and other molecules have been developed that bind the virus, no therapeutic has shown the efficacy needed for FDA approval. Here, a library of 50 peptide based ligands that bind the glycoprotein of the Zaire Ebola virus (GP) were developed. Using whole virus screening of vesicular stomatitis virus pseudotyped with GP, low affinity peptides were identified for ligand construction. In depth analysis showed that two of the peptide based molecules bound the Zaire GP with <100 nM KD. One of these two ligands was blocked by a known neutralizing mAb, 2G4, and showed cross-reactivity to the Sudan GP. This work presents ligands with promise for therapeutic applications across multiple variants of the Ebola virus.
ContributorsRabinowitz, Joshua Avraam (Author) / Diehnelt, Chris (Thesis director) / Johnston, Stephen (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The parameters of microwave-assisted acid hydrolysis (MAAH) and 1H NMR highly affect the quantitative analysis of protein hydrolysates. Microwave-induction source, NMR spectral resolution, and data analysis are key parameters in the nuclear magnetic resonance – amino acid analysis (NMR-AAA) workflow where errors accrue due to lack of an optimized protocol. Hen egg white lysozyme was hydrolyzed using an 800W domestic microwave oven for varying time points between 10-25 minutes, showing minimal protein hydrolysis after extended time periods. Studies on paramagnetic doping with varying amounts of gadolinium chloride for increased NMR resolution resulted in little T1 reduction in a majority of amino acids and resulted in significant line broadening in concentrations above 1µM. The use of the BAYESIL analysis tool with HOD suppressed 1H-NMR spectra resulted in misplaced template peaks and errors greater than 1% for 10 of 13 profiled amino acids with the highest error being 7.6% (Thr). Comparatively, Chenomx NMR Suite (v7.1) analysis resulted in errors of less than 1% for 9 of 13 profiled amino acids with a highest error value of 3.6% (Lys). Using the optimized protocol, hen egg white lysozyme C was identified at rank 1 with a score of 64 in a Gallus gallus species wide AACompIdent search. This technique reduces error associated with sample handling relative to previously used amino acid analysis (AAA) protocols and requires no derivatization or additional processing of the sample prior to analysis.
ContributorsJordan, Jacob Smith (Author) / Yarger, Jeffery (Thesis director) / Van Horn, Wade (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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
Gle1 is an mRNP export mediator with major activity localized to the nuclear pore complex in eukaryotic cells. The protein's high preservation across vast phylogenetic distances allows us to approximate research on the properties of yeast Gle1 (yGle1) with those of human Gle1 (hGle1). Research at Vanderbilt University in 2016, which provides the research basis of this thesis, suggests that the coiled-coil domain of yGle1 is best crystallized in dicationic aqueous conditions of pH ~8.0 and 10\u201420% PEG 8000. Further exploration of crystallizable microconditions revealed a favorability toward lower pH and lower PEG concentration. Following the discovery of the protein's native crystallography conditions, a comprehensive meta-analysis of scientific literature on Gle1 was conducted on the association of Gle1 mutations with neuron disease.
ContributorsGaetano, Philip Pasquale (Author) / Foy, Joseph (Thesis director) / Dawson, T. Renee (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Predicting the binding sites of proteins has historically relied on the determination of protein structural data. However, the ability to utilize binding data obtained from a simple assay and computationally make the same predictions using only sequence information would be more efficient, both in time and resources. The purpose of this study was to evaluate the effectiveness of an algorithm developed to predict regions of high-binding on proteins as it applies to determining the regions of interaction between binding partners. This approach was applied to tumor necrosis factor alpha (TNFα), its receptor TNFR2, programmed cell death protein-1 (PD-1), and one of its ligand PD-L1. The algorithms applied accurately predicted the binding region between TNFα and TNFR2 in which the interacting residues are sequential on TNFα, however failed to predict discontinuous regions of binding as accurately. The interface of PD-1 and PD-L1 contained continuous residues interacting with each other, however this region was predicted to bind weaker than the regions on the external portions of the molecules. Limitations of this approach include use of a linear search window (resulting in inability to predict discontinuous binding residues), and the use of proteins with unnaturally exposed regions, in the case of PD-1 and PD-L1 (resulting in observed interactions which would not occur normally). However, this method was overall very effective in utilizing the available information to make accurate predictions. The use of the microarray to obtain binding information and a computer algorithm to analyze is a versatile tool capable of being adapted to refine accuracy.
ContributorsBrooks, Meilia Catherine (Author) / Woodbury, Neal (Thesis director) / Diehnelt, Chris (Committee member) / Ghirlanda, Giovanna (Committee member) / Department of Psychology (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05