Matching Items (3)
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- All Subjects: Crystallography
- Creators: Chiu, Po-Lin
Description
The human gastrin receptor (CCKBR or CCK2R) is a class A G protein-coupled receptor (GPCR) found throughout the central nervous system, stomach, and a variety of cancer cells. CCK2R is implicated in the regulation of biological processes, including anxiety, satiety, arousal, analgesia, psychosis, and cancer cell growth and proliferation. While CCK2R is an attractive drug target, few drugs have managed to effectively target the receptor, and none have been brought to market. Contributory to this is the lack of high-resolution crystal structure capable of elucidating the binding regions of CCK2R to streamlining drug screening. While GPCRs are not amenable to traditional structural analysis methodologies, the advent of lipidic cubic phase (LCP) crystallography and serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs), has extended the applicability of X-ray crystallography to these integral membrane proteins. LCP-SFX depends on optimizing the protein of interest for extraction, purification, and crystallization. Here we report our findings regarding the optimization of CCK2R suggesting the synergistic relationship between N-terminal truncations and the insertion of a fusion protein along ICL3, in addition to a 30-residue truncation of the C-terminus. Samples were expressed in Sf9 insect cells using a Bac-to-Bac baculovirus expression system, extracted using n-Dodecyl-β-D-Maltoside detergent, and purified via TALON immobilized metal-ion affinity chromatography. The constructs were characterized via SDS-PAGE, Western blot, and size exclusion chromatography. These findings demonstrate the improvements to CCK2R’s crystallographic amenability upon these modifications, however significant improvements must be made prior to crystallization trials. Future work will involve screening C-terminal truncations, thermostabilizing point mutations, and co-crystallizing ligands. Ideally this investigation will serve as a model for future CCK2R structural analysis and contribute to a heightened interest in CCK2R as a therapeutic target.
ContributorsStevens, Alexander Wade (Author) / Liu, Wei (Thesis director) / Chiu, Po-Lin (Committee member) / Mills, Jeremy (Committee member) / School of Human Evolution & Social Change (Contributor) / School of Molecular Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data. Additionally, differential and specific cholesterol binding in the CCK receptor subfamily was observed while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. Mutation of this conserved CRAC sequence of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase, however, as most techniques, it has limitations. Using an optimized SFX experimental setup in a helium atmosphere we determined the room temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution and compared it with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, we demonstrated the capability of utilizing high XFEL beam transmissions, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete data set.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
ContributorsGeiger, James (Author) / Liu, Wei (Thesis advisor) / Mills, Jeremy (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020
Description
Macromolecular structural biology advances the understanding of protein function through the structure-function relationship for applications to scientific challenges like energy and medicine. The proteins described in these studies have applications to medicine as targets for therapeutic drug design. By understanding the mechanisms and dynamics of these proteins, therapeutics can be designed and optimized based on their unique structural characteristics. This can create new, focused therapeutics for the treatment of diseases with increased specificity — which translates to greater efficacy and fewer off-target effects. Many of the structures generated for this purpose are “static” in nature, meaning the protein is observed like a still-frame photograph; however, the use of time-resolved techniques is allowing for greater understanding of the dynamic and flexible nature of proteins. This work advances understanding the dynamics of the medically relevant proteins NendoU and Taspase1 using serial crystallography to establish conditions for time-resolved, mix-and-inject crystallographic studies.
ContributorsJernigan, Rebecca Jeanne (Author) / Fromme, Petra (Thesis advisor) / Hansen, Debra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Hogue, Brenda (Committee member) / Arizona State University (Publisher)
Created2022