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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
ABSTRACT
Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation.
In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work.
In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro.
Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation.
In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work.
In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro.
ContributorsMorales Betanzos, Carlos (Author) / LaBaer, Joshua (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Description
An animal's ability to produce protein-based silk materials has evolved independently in many different arthropod lineages, satisfying various ecological necessities. However, regardless of their wide range of uses and their potential industrial and biomedical applications, advanced knowledge on the molecular structure of silk biopolymers is largely limited to those produced by spiders (order Araneae) and silkworms (order Lepidoptera). This thesis provides an in-depth molecular-level characterization of silk fibers produced by two vastly different insects: the caddisfly larvae (order Trichoptera) and the webspinner (order Embioptera).
The molecular structure of caddisfly larval silk from the species Hesperophylax consimilis was characterized using solid-state nuclear magnetic resonance (ss-NMR) and Wide Angle X-ray Diffraction (WAXD) techniques. This insect, which typically dwells in freshwater riverbeds and streams, uses silk fibers as a strong and sticky nanoadhesive material to construct cocoons and cases out available debris. Conformation-sensitive 13C chemical shifts and 31P chemical shift anisotropy (CSA) information strongly support a unique protein motif in which phosphorylated serine- rich repeats (pSX)4 complex with di- and trivalent cations to form rigid nanocrystalline β-sheets. Additionally, it is illustrated through 31P NMR and WAXD data that these nanocrystalline structures can be reversibly formed, and depend entirely on the presence of the stabilizing cations.
Nanofiber silks produced by webspinners (order Embioptera) were also studied herein. This work addresses discrepancies in the literature regarding fiber diameters and tensile properties, revealing that the nanofibers are about 100 nm in diameter, and are stronger (around 500 MPa mean ultimate stress) than previous works suggested. Fourier-transform Infrared Spectroscopy (FT-IR), NMR and WAXD results find that approximately 70% of the highly repetitive glycine- and serine-rich protein core is composed of β-sheet nanocrystalline structures. In addition, FT-IR and Gas-chromatography mass spectroscopy (GC-MS) data revealed a hydrophobic surface coating rich in long-chain lipids. The effect of this surface coating was studied with contact angle techniques; it is shown that the silk sheets are extremely hydrophobic, yet due to the microstructural and nanostructural details of the silk surface, are surprisingly adhesive to water.
The molecular structure of caddisfly larval silk from the species Hesperophylax consimilis was characterized using solid-state nuclear magnetic resonance (ss-NMR) and Wide Angle X-ray Diffraction (WAXD) techniques. This insect, which typically dwells in freshwater riverbeds and streams, uses silk fibers as a strong and sticky nanoadhesive material to construct cocoons and cases out available debris. Conformation-sensitive 13C chemical shifts and 31P chemical shift anisotropy (CSA) information strongly support a unique protein motif in which phosphorylated serine- rich repeats (pSX)4 complex with di- and trivalent cations to form rigid nanocrystalline β-sheets. Additionally, it is illustrated through 31P NMR and WAXD data that these nanocrystalline structures can be reversibly formed, and depend entirely on the presence of the stabilizing cations.
Nanofiber silks produced by webspinners (order Embioptera) were also studied herein. This work addresses discrepancies in the literature regarding fiber diameters and tensile properties, revealing that the nanofibers are about 100 nm in diameter, and are stronger (around 500 MPa mean ultimate stress) than previous works suggested. Fourier-transform Infrared Spectroscopy (FT-IR), NMR and WAXD results find that approximately 70% of the highly repetitive glycine- and serine-rich protein core is composed of β-sheet nanocrystalline structures. In addition, FT-IR and Gas-chromatography mass spectroscopy (GC-MS) data revealed a hydrophobic surface coating rich in long-chain lipids. The effect of this surface coating was studied with contact angle techniques; it is shown that the silk sheets are extremely hydrophobic, yet due to the microstructural and nanostructural details of the silk surface, are surprisingly adhesive to water.
ContributorsAddison, John Bennett (Author) / Yarger, Jeffery L (Thesis advisor) / Holland, Gregory P (Thesis advisor) / Wang, Xu (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2014
Description
The small mitogenic cytokine Pleiotrophin (PTN) is well-known for its roles in
tissue growth, development, and repair. First isolated from neuronal tissues, much interest in this protein resides in development of the central nervous system and neuronal regeneration. Owning to its role in growth, development and its ability to promote angiogenesis and metastasis, PTN’s overexpression in cancers such as glioblastoma, has become the focal point of much research. Many of the receptors through which PTN acts contain glycosaminoglycans (GAGs), through which PTN binds. Thus, understanding the atomistic detail of PTN’s architecture and interaction with GAG chains is of significant importance in elucidating its functional role in growth and malignancy of biological tissues, as well as in neural development and progression of other diseases. Herein the first solution state structure of PTN was solved via nuclear magnetic resonance (NMR), with extensive characterization of its ability to bind GAG. Structurally, PTN consists of two -sheet domains connected by a short flexible linker, and flanked by long flexible termini. Broad distribution of positively charged amino acids in the protein’s sequence yields highly basic surfaces on the -sheet domains as well as highly cationic termini. With GAG chains themselves being linear anionic polymers, all interactions between these sugars and PTN are most exclusively driven through the electrostatic interactions between them, with no discernable specificity for GAG types. Moreover, this binding event is coordinated mostly through basic patches located in the C-Terminal domain (CTD). Although the flexible C- terminus has been shown to play a significant role in receptor binding, data here also reveal an adaptability of PTN to maintain high affinity interactions through its structured domains
when termini are removed. Additionally, analysis of binding information revealed for the first time the presence of a secondary GAG binding site within PTN. It is shown that PTN’s CTD constitutes the major binding site, while the N-terminal domain (NTD) contains the much weaker secondary site. Finally, compilation of high-resolution data containing the atomistic detail of PTN’s interaction with GAG provided the information necessary to produce the highest accuracy model to date of the PTN-GAG complex. Taken together, these findings provide means for specific targeting of this mitogenic cytokine in a wide array of biological applications.
tissue growth, development, and repair. First isolated from neuronal tissues, much interest in this protein resides in development of the central nervous system and neuronal regeneration. Owning to its role in growth, development and its ability to promote angiogenesis and metastasis, PTN’s overexpression in cancers such as glioblastoma, has become the focal point of much research. Many of the receptors through which PTN acts contain glycosaminoglycans (GAGs), through which PTN binds. Thus, understanding the atomistic detail of PTN’s architecture and interaction with GAG chains is of significant importance in elucidating its functional role in growth and malignancy of biological tissues, as well as in neural development and progression of other diseases. Herein the first solution state structure of PTN was solved via nuclear magnetic resonance (NMR), with extensive characterization of its ability to bind GAG. Structurally, PTN consists of two -sheet domains connected by a short flexible linker, and flanked by long flexible termini. Broad distribution of positively charged amino acids in the protein’s sequence yields highly basic surfaces on the -sheet domains as well as highly cationic termini. With GAG chains themselves being linear anionic polymers, all interactions between these sugars and PTN are most exclusively driven through the electrostatic interactions between them, with no discernable specificity for GAG types. Moreover, this binding event is coordinated mostly through basic patches located in the C-Terminal domain (CTD). Although the flexible C- terminus has been shown to play a significant role in receptor binding, data here also reveal an adaptability of PTN to maintain high affinity interactions through its structured domains
when termini are removed. Additionally, analysis of binding information revealed for the first time the presence of a secondary GAG binding site within PTN. It is shown that PTN’s CTD constitutes the major binding site, while the N-terminal domain (NTD) contains the much weaker secondary site. Finally, compilation of high-resolution data containing the atomistic detail of PTN’s interaction with GAG provided the information necessary to produce the highest accuracy model to date of the PTN-GAG complex. Taken together, these findings provide means for specific targeting of this mitogenic cytokine in a wide array of biological applications.
ContributorsRyan, Eathen (Author) / Wang, Xu (Thesis advisor) / Yarger, Jeffery (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
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
Spatial resolved detection and quantification of ribonucleic acid (RNA) molecules in single cell is crucial for the understanding of inherent biological issues, like mechanism of gene regulation or the development and maintenance of cell fate. Conventional methods for single cell RNA profiling, like single-cell RNA sequencing (scRNA-seq) or single-molecule fluorescent in situ hybridization (smFISH), suffer either from the loss of spatial information or the low detection throughput. In order to advance single-cell analysis, new approaches need to be developed with the ability to perform high-throughput detection while preserving spatial information of the subcellular location of target RNA molecules.
Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.
Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.
ContributorsXiao, Lu, Ph.D (Author) / Guo, Jia (Thesis advisor) / Wang, Xu (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2019
Description
The major goal of this large project is to develop a Recognition Tunneling Nanopore (RTP) device that will be used for determining the structure of glycosaminoglycans (GAGs). The RTP device is composed of a recognition tunneling junction that is embedded in a nanopore. In order to translocate the GAG molecule through the nanopore, researchers have designed a scheme in which the GAG molecule of interest will be attached to the 5’ end of a DNA primer (figure 1) and the DNA primer will be extended by a biotinylated Φ29 DNA polymerase that is anchored in the nanoslit using streptavidin. This research project specifically is part of a larger project with the main goal of comparing the activity of the wild-type Φ29 DNA polymerase which I have expressed and purified with the mutated Φ29 DNA polymerase devoid of 3’ - 5’ exonuclease activity which was made by Dr. Deng.
ContributorsDadkhah Tirani, Farbod (Author) / Wang, Xu (Thesis director) / Zhang, Peiming (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05