This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Even with adequate expression levels, the study of membrane proteins is limited by difficulties in extracting and sometimes refolding the proteins. We will therefore establish a strategy for choosing detergents and lipids optimal for purification, solubilization, and stabilization of native structure. This will include studies of the properties, particularly phase behavior, of the surfactant systems themselves, as well as of their behavior in the presence of membrane proteins. In addition to guiding refolding strategies, this information will be useful in its own right. Our results will provide increased knowledge of the mechanism of membrane protein insertion, folding, and interactions with detergents and lipids, and will facilitate production of significant quantities of active integral membrane proteins. The goal of this work is to engineer a heterologous GPCR expression system in the yeast S. cerevisiae in order to obtain high levels of purified, functional G-protein coupled receptors for biophysical characterization and high-resolution structure determination. To achieve this goal, we seek to better characterize S. cerevisiae as a host system by carefully examining receptor trafficking and folding, as well as intracellular stress responses that may be triggered during expression. By identifying the conditions that allow for high levels of functional expression of GPCRs in yeast, this system will serve as a more desirable host to accelerate mammalian GPCR over-expression and biophysical characterization. The goals of this project are listed below: -Characterize the human adenosine A2a receptor using biophysical methods -Engineer S. cerevisiae to express milligram amounts of active mammalian GPCRs This subproject also covers topics originally included in SPID 0021 but incorporated in SPID 0020 earlier in the grant. This research involves establishing a strategy for choosing detergents and lipids optimal for purification, solubilization, and stabilization of native structure in membrane proteins. The phase behavior of surfactant solutions containing additives used in protein crystallization will also be investigated in order to guide efforts to crystallize membrane proteins more reliably.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Exploratory Grants (P20)
Project #
5P20RR015588-10
Application #
7959538
Study Section
Special Emphasis Panel (ZRR1-RI-8 (01))
Project Start
2009-06-01
Project End
2010-05-31
Budget Start
2009-06-01
Budget End
2010-05-31
Support Year
10
Fiscal Year
2009
Total Cost
$384,732
Indirect Cost
Name
University of Delaware
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
059007500
City
Newark
State
DE
Country
United States
Zip Code
19716
Li, Linqing; Stiadle, Jeanna M; Levendoski, Elizabeth E et al. (2018) Biocompatibility of injectable resilin-based hydrogels. J Biomed Mater Res A 106:2229-2242
Bathala, Pradeepthi; Fereshteh, Zeinab; Li, Kun et al. (2018) Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility. Mol Hum Reprod 24:143-157
Olli, Kristine E; Li, Kun; Galileo, Deni S et al. (2018) Plasma membrane calcium ATPase 4 (PMCA4) co-ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity. J Cell Physiol 233:11-22
Wu, Kathie Z; Li, Kun; Galileo, Deni S et al. (2017) Junctional adhesion molecule A: expression in the murine epididymal tract and accessory organs and acquisition by maturing sperm. Mol Hum Reprod 23:132-140
Li, Linqing; Stiadle, Jeanna M; Lau, Hang K et al. (2016) Tissue engineering-based therapeutic strategies for vocal fold repair and regeneration. Biomaterials 108:91-110
Martin-DeLeon, Patricia Anastasia (2016) Uterosomes: Exosomal cargo during the estrus cycle and interaction with sperm. Front Biosci (Schol Ed) 8:115-22
Al-Dossary, Amal A; Bathala, Pradeepthi; Caplan, Jeffrey L et al. (2015) Oviductosome-Sperm Membrane Interaction in Cargo Delivery: DETECTION OF FUSION AND UNDERLYING MOLECULAR PLAYERS USING THREE-DIMENSIONAL SUPER-RESOLUTION STRUCTURED ILLUMINATION MICROSCOPY (SR-SIM). J Biol Chem 290:17710-23
Monillas, Elizabeth S; Caplan, Jeffrey L; Thévenin, Anastasia F et al. (2015) Oligomeric state regulated trafficking of human platelet-activating factor acetylhydrolase type-II. Biochim Biophys Acta 1854:469-75
Andrews, Rachel E; Galileo, Deni S; Martin-DeLeon, Patricia A (2015) Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase. Mol Hum Reprod 21:832-43
Hu, Yuan; Sinha, Sudipta Kumar; Patel, Sandeep (2015) Investigating Hydrophilic Pores in Model Lipid Bilayers Using Molecular Simulations: Correlating Bilayer Properties with Pore-Formation Thermodynamics. Langmuir 31:6615-31

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