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Probing the structure of Calbinding D28k using constraints from intramolecular cross-links and molecular modeling

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Probing the structure of Calbinding D28k using constraints from intramolecular cross-links and molecular modeling

Yeung, David Yong-Hoi (2004) Probing the structure of Calbinding D28k using constraints from intramolecular cross-links and molecular modeling. Masters thesis, Concordia University.

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Abstract

Calbindin D28k is a calcium binding protein of unknown structure. It is believed to be composed of six EF-hand subdomains (helix-loop-helix motifs), which is supported in part by NMR analysis of a recombinant fragment containing the first and second EF hands. A recent study demonstrated the structural characterization of fibroblast growth factor using sequence alignment and distance constraints based on several identified intramolecular chemical cross-links. A similar approach is applied in this thesis to structurally characterize human calbindin D 28k . Reactions of the protein with a bis(sulfosuccinimidyl) suberate, a lysine specific cross-linker, yields primarily protein modified with a single cross-linker. Identified cross-linked peptides in the MALDI mass fingerprints of tryptic digests of modified calbindin D 28k impose distance constraints between pairs of lysine residues. Applying these constraints, hypothetical structures of the protein were constructed. Of particular interest is the orientation of the EF-hand subdomains with respect to each other. Of the 103 configurations considered, only 14 respected the distance constraints. One of these 14 structures was selected to demonstrate the use of molecular mechanics for structure refinement. Cross-linking of myoglobin, a protein of known structure, was first performed to evaluate the experimental method. Because the calcium loaded form of calbindin D 28k was studied, the calcium coordination spheres of four of its EF-hands were investigated using molecular modeling. Additionally, the expression and purification of recombinant human calbindin D 28k is described.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Yeung, David Yong-Hoi
Pagination:x, 109 leaves : ill. ; 29 cm. + 1 CD-ROM (col. ; 4 3/4 in.)
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry and Biochemistry
Date:2004
Thesis Supervisor(s):English, Ann M and Peslherbe, Gilles H
ID Code:8147
Deposited By:Concordia University Libraries
Deposited On:18 Aug 2011 14:16
Last Modified:18 Aug 2011 15:45
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