Matching Items (5)
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- All Subjects: Membrane Proteins
- Creators: Liu, Wei
- Creators: Wang, Xu
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
The understanding of normal human physiology and disease pathogenesis shows great promise for progress with increasing ability to profile genomic loci and transcripts in single cells in situ. Using biorthogonal cleavable fluorescent oligonucleotides, a highly multiplexed single-cell in situ RNA and DNA analysis is reported. In this report, azide-based cleavable linker connects oligonucleotides to fluorophores to show nucleic acids through in situ hybridization. Post-imaging, the fluorophores are effectively cleaved off in half an hour without loss of RNA or DNA integrity. Through multiple cycles of hybridization, imaging, and cleavage this approach proves to quantify thousands of different RNA species or genomic loci because of single-molecule sensitivity in single cells in situ. Different nucleic acids can be imaged by shown by multi-color staining in each hybridization cycle, and that multiple hybridization cycles can be run on the same specimen. It is shown that in situ analysis of DNA, RNA and protein can be accomplished using both cleavable fluorescent antibodies and oligonucleotides. The highly multiplexed imaging platforms will have the potential for wide applications in both systems biology and biomedical research. Thus, proving to be cost effective and time effective.
ContributorsSamuel, Adam David (Author) / Guo, Jia (Thesis director) / Liu, Wei (Committee member) / Wang, Xu (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
G protein-coupled receptors, or GPCRs, are receptors located within the membrane of cells that elicit a wide array of cellular responses through their interactions with G proteins. Recent advances in the use of lipid cubic phase (LCP) for the crystallization of GPCRs, as well as increased knowledge of techniques to improve receptor stability, have led to a large increase in the number of available GPCR structures, despite historic difficulties. This project is focused on the histamine family of receptors, which are Class A GPCRs that are involved in the body’s allergic and inflammatory responses. In particular, the goal of this project was to design, express, and purify histamine receptors with the ultimate goal of crystallization. Successive rounds of optimization included the use of recombinant DNA techniques in E.coli to truncate sections of the proteins and the insertion of several fusion partner proteins to improve receptor expression and stability. All constructs were expressed in a Bac-to-Bac baculovirus expression system using Sf9 insect cells, solubilized using n-Dodecyl-β-D-Maltoside (DDM), and purified using immobilized metal affinity chromatography. Constructs were then analyzed by SDS-Page, Western blot, and size-exclusion chromatography to determine their presence, purity, and homogeneity. Along with their expression data from insect cells, the most stable and homogeneous construct from each round was used to design successive optimizations. After 3 rounds of construct design for each receptor, much work remains to produce a stable sample that has the potential to crystallize. Future work includes further optimization of the insertion site of the fusion proteins, ligand screening for co-crystallization, optimization of purification conditions, and screening of potential thermostabilizing point mutations. Success in solving a structure will allow for a more detailed understanding of the receptor function in addition to its vital use in rational drug discovery.
ContributorsCosgrove, Steven Andrew (Author) / Liu, Wei (Thesis director) / Mills, Jeremy (Committee member) / Mazor, Yuval (Committee member) / W. P. Carey School of Business (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
In the past decade, technological breakthroughs have facilitated structure determination of so many difficult-to-study membrane protein targets. In this thesis research, three techniques were investigated to enable the structural determination of such challenging targets, polychromatic pink-beam serial crystallography with high-viscous sample, lipidic cubic phase (LCP)-based microcrystal electron diffraction (MicroED), and single-particle cryogenic electron microscopy targeting (cryoEM).
Inspired by the successful serial crystallography (SX) experiment at a synchrotron radiation source, it is first-time equipping the high-viscosity injector to X-ray fluxes increased at 100 times by a moderate increased in bandwidth to perform the pink beam SX experiments. The structure of proteinase K (PK) was determined to 1.8 Å resolution with 4 consecutive 100 ps X-ray pink beam pulse exposures. The structure of human A2A adenosine receptor (A2AAR) reached to a 4.2 Å resolution using 24 consecutive X-ray pink beam pulse exposures. It has proven the feasibility to utilize such storage-ring synchrotron sources complemented to serial femtosecond crystallography, presenting new opportunities for microcrystallography and the time-resolved experiments.
As an alternative approach to serial femtosecond crystallography, a novel protocol was developed to combine the lipidic cubic phase crystallization approach and microED strategy and solved the structure from LCP-embedded proteinase K microcrystals with the comparable high resolution to conventional crystallographic method.
It cannot be neglected that only very few portions of membrane proteins were able to be successfully crystallized for structure determination. Single particle cryoEM method allows the structural studies from protein molecules detour away from crystallization. An atomic resolution structure of the β1-AR bound with agonist in complex with Gs protein, with particle size of less than 200 kDa, was determined by cryoEM, reaching to an atomic resolution of 3.8 Å. The complex structure captured a fully active conformation and revealed the important mechanisms of how the agonist bound receptor activated Gs protein.
These technological developments provide more opportunities to the structural biology community to discover mechanisms underlying such complicated machinery network, which would eventually benefit the structure-based drug discovery.
Inspired by the successful serial crystallography (SX) experiment at a synchrotron radiation source, it is first-time equipping the high-viscosity injector to X-ray fluxes increased at 100 times by a moderate increased in bandwidth to perform the pink beam SX experiments. The structure of proteinase K (PK) was determined to 1.8 Å resolution with 4 consecutive 100 ps X-ray pink beam pulse exposures. The structure of human A2A adenosine receptor (A2AAR) reached to a 4.2 Å resolution using 24 consecutive X-ray pink beam pulse exposures. It has proven the feasibility to utilize such storage-ring synchrotron sources complemented to serial femtosecond crystallography, presenting new opportunities for microcrystallography and the time-resolved experiments.
As an alternative approach to serial femtosecond crystallography, a novel protocol was developed to combine the lipidic cubic phase crystallization approach and microED strategy and solved the structure from LCP-embedded proteinase K microcrystals with the comparable high resolution to conventional crystallographic method.
It cannot be neglected that only very few portions of membrane proteins were able to be successfully crystallized for structure determination. Single particle cryoEM method allows the structural studies from protein molecules detour away from crystallization. An atomic resolution structure of the β1-AR bound with agonist in complex with Gs protein, with particle size of less than 200 kDa, was determined by cryoEM, reaching to an atomic resolution of 3.8 Å. The complex structure captured a fully active conformation and revealed the important mechanisms of how the agonist bound receptor activated Gs protein.
These technological developments provide more opportunities to the structural biology community to discover mechanisms underlying such complicated machinery network, which would eventually benefit the structure-based drug discovery.
ContributorsZhu, Lan, Ph.D (Author) / Liu, Wei (Thesis advisor) / Mills, Jeremy (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2019
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