The Glycine receptor (GlyR) is the major inhibitory neurotransmitter receptor in the spinal cord and brainstem. Dysfunction of GlyR causes hyperekplexia, a neurological disease characterized by an excessive startle response. However, limited structural information about this physiologically important receptor is available. Therefore, direct structural analyses at high resolution of truncated ligand binding domains, and possibly full-length GlyR, are required for further understanding of this important neurotransmitter receptor.
This study is focused on purifying and characterizing glycine binding protein (GlyBP), a mutant form of the ligand binding domain of the GlyR, in which two hydrophobic loops were replaced with corresponding hydrophilic residues in AChBP. GlyBP was overexpressed in Sf9 insect cells. GlyBP was found in both cytosolic and membrane-bound fractions after subcellular fractionation. The cytosolic fraction was misfolded. In contrast, the membrane-bound form is functional as shown by its ability to reversibly bind to 2-aminostrychnine resin. After affinity purification, membrane-bound GlyBP could be isolated in an aqueous form and a membrane-associated vesicular form. Radiolabeled binding assays showed both forms of GlyBP retained abilities to bind to its ligands, with affinities comparable to those of full-length GlyR. Furthermore, studies using chemical crosslinking, light scattering and luminescence resonance energy transfer (LRET) showed that both forms of GlyBP are oligomeric, and are very likely pentameric. The LRET studies also showed GlyBP undergoes conformational changes upon glycine binding equivalent to changes in full-length GlyR. Further studies using chemical crosslinking coupled with mass spectrometry were conducted to probe the low resolution three-dimensional structure and inter-subunit interactions. A number of intramolecular and/or intermolecular Lys-Lys crosslinks were identified. Those crosslinks provided useful information about protein folding and validated our computationally-derived model of GlyBP.
Results from this study indicate that GlyBP adopts a native-like structure and is a structural and functional homolog of the extracellular domain of GlyRs and other members in Cys-loop receptor family. Further detailed structural studies will lead to further understanding of function of the ligand binding domain of GlyRs. In addition, efforts on resolving a high-resolution structure of GlyBP might result in detailed structural information about this physiologically important receptor and also other Cys-loop receptors.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12182007-154915 |
| Date | 20 December 2007 |
| Creators | Liu, Zhenyu |
| Contributors | Yan Xu, Marc J. Glucksman, Jon W. Johnson, Maria Kurnikova, Gonzalo E. Torres |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-12182007-154915/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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