Electronic Theses and Dissertations

Identifier

245

Date

2011-04-18

Document Type

Dissertation (Campus Access Only)

Degree Name

Doctor of Philosophy

Major

Chemistry

Concentration

Organic Chemistry

Committee Chair

Abby L. Parrill

Committee Member

John K. Young

Committee Member

Daniel L. Baker

Committee Member

Theodore J. Burkey

Abstract

Studies suggest that the extracellular loops (ECLs) of the lysophosphatidic acid (LPA) receptors are involved in antagonist binding. ECL2 is known to be involved in conformational flexibility and ligand binding in other Family A GPCR, but which has not been determined in the LPA receptors. Abnormal LPA activity is associated with cancer. Contributions to the disease mechanism involve the overproduction of LPA by autotaxin (ATX) and/or the overexpression of LPA receptors. A possible means of intervening consists of blocking LPA from activating the aberrant LPA receptor(s). Development of a structure-based pharmacophore model of the LPA1 receptor would benefit from more accurate structural information about the ECL2. A fragment-based, structural study of the LPA1 receptor ECL2 using NMR spectroscopy can be used to obtain an atomic resolution structure. Mimetics of the ECL2 were designed to prevent random folding and precipitation during NMR experiments. The LPA1 receptor sequences of the transmembrane helices flanking the ECL2 were replaced with coiled-coil sequences for favorable folding in solution. Four loop mimetics were designed. Two loop mimetics consisted of different lengths of coiled-coil sequences from protein kinase PKN/PRK1 (pdb entry 1CXZ), and another mimetic in which mutations were made to this design. The last design consisted of a de novo coiled-coil with a sequence based on protein folding theory. The structure of the loop mimetics were evaluated using circular dichroism (CD) and NMR spectroscopy. The ECL2 short mimetic resulted in mostly random folding in solution and required the elongation of the coiled-coil sequences found in the ECL2 long mimetic design, which was stable at low protein concentrations. However, higher concentrations of the ECL2 long mimetic resulted in structural disfiguration, which could not be rectified by the chosen mutations to this design. The ECL2 de novo mimetic resulted in favorable protein folding without structural disfiguration based on 2D/3D NMR techniques. Backbone assignments were made for this design. Analysis of the backbone chemical shifts showed significant secondary structure along appropriate stretches of sequence. So, the ECL2 de novo mimetic resulted in favorable folding suitable for obtaining a high resolution protein structure.

Comments

Data is provided by the student.

Library Comment

Dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.

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