Show simple item record

dc.contributor.authorFralish, Gregory Brian
dc.date.accessioned2014-03-03T20:19:45Z
dc.date.available2014-03-03T20:19:45Z
dc.date.issued2002-12
dc.identifier.otherfralish_gregory_b_200212_phd
dc.identifier.urihttp://purl.galileo.usg.edu/uga_etd/fralish_gregory_b_200212_phd
dc.identifier.urihttp://hdl.handle.net/10724/20558
dc.description.abstractHuman chorionic gonadotropin (hCG) is critical to the maintenance of pregnancy and male sexual differentiation. It is a member of the family of glycoprotein hormones, which includes luteinizing hormone (LH), follicle stimulating hormone and thyroid stimulating hormone. This family of hormones is characterized by a heterodimeric structure, consisting of a common á -subunit and a hormone specific â -subunit. hCG (and LH) bind to their common receptor, LHR, with high affinity to elicit its biological response(s). LHR is a G protein-coupled receptor with a large, leucine-rich repeat (LRR)-containing extracellular domain (ECD) that is responsible for the high affinity contacts with hormone. Since there is little structural data available on the receptor, our understanding of the binding of hCG and the LHR ECD is limited to indirect observations and models. The goal of this dissertation is to provide direct structural data for this complex system using fusion proteins of the heterodimeric hormone and hormone-receptor complexes. Therefore, an expression and purification system was developed for the two single-chain hormones, YhCG1 (â -á ), which has similar bioactivity to native hCG, and YhCG3 (á -â ), which has altered binding properties. The purified hormones were characterized structurally using circular dichroism (CD) and limited proteolysis. These studies have revealed that there is a remarkable variability in the structures of the different hormones. Nevertheless, the proteins can functionally interact and activate the receptor suggesting an unexpected conformational tolerance for a productive hormone-receptor interaction. Furthermore, an expression and purification system was developed for three hormone-receptor ECD fusion proteins. By expressing the ligand tethered to the full-length receptor ectodomain (YECD), expression levels of the complex were improved 20-fold over those previously reported, thus making possible detailed structural studies. This fusion complex was shown to be functional, as it can bind hCG with high affinity. Using CD spectroscopy, YECD revealed that the ECD contains between 18-20% á helix and 30% â -strand. These data provide structural evidence in support of the proposed homology model, and suggest that 8 or 9 LRRs are present in the ECD. Further, they provide proof of principle for using ligand receptor fusions to express functional complexes. The structural characterization of two C-terminal truncations of YECD demonstrated a repeating motif in the ECD. Furthermore, amino acid residues in the C-terminal region of the ECD are important in the overall stabilization of the ECD, an intriguing observation considering this region’s emerging role as a signal modulator.
dc.languageeng
dc.publisheruga
dc.rightspublic
dc.subjecthuman chorionic gonadotropin
dc.subjectLH receptor
dc.subjectbaculovirus expression
dc.subjectextracellular domain
dc.subjectsingle-chain gonadotropin
dc.subjectyoked gonadotropin
dc.titleTowards an understanding of luteinizing hormone receptor binding
dc.typeDissertation
dc.description.degreePhD
dc.description.departmentBiochemistry and Molecular Biology
dc.description.majorBiochemistry and Molecular Biology
dc.description.advisorDavid Puett
dc.description.committeeDavid Puett
dc.description.committeeBi-Cheng Wang
dc.description.committeeJames Travis
dc.description.committeeKojo Mensa-Wilmot
dc.description.committeeClaiborne Glover


Files in this item

FilesSizeFormatView

There are no files associated with this item.

This item appears in the following Collection(s)

Show simple item record