Theses and Dissertations - Department of Chemistry & Biochemistry

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Browsing Theses and Dissertations - Department of Chemistry & Biochemistry by Subject "Chemistry, Biochemistry"

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    Low-molecular-weight chromium-binding substance: advanced studies from aves to human
    (University of Alabama Libraries, 2009) Chen, Yuan; Vincent, John B.; University of Alabama Tuscaloosa
    Chromium has been observed to play a role in maintaining proper carbohydrate and lipid metabolism of mammals. One of the potentially biological active forms of chromium in vitro is low-molecular-weight chromium-binding substance (LMWCr), which has been proposed to amplify the insulin cascade by binding with insulin receptor. LWMCr is a small bio-molecule (<1500 Da) containing a carboxylate-rich polypeptide with four bound chromic ions. LMWCr's (according to its amino acid composition and mass spectrum) were successfully isolated from chicken and alligator livers, as well as from human urine, using a modified method. The extreme hydrophilicity of the peptide and the tightly bound Cr(III) are two major hurdles to produce a stable end-product for mass spectrometry (MS) or high-performance liquid chromatography (HPLC) analysis. Treating bovine LMWCr with trifluoroacetic acid and application to a graphite powder micro-column was used to generate a heptapeptide fragment, and the peptide sequence was analyzed by mass spectrometry (MS) and tandem MS (MS/MS). Two candidate sequences, EEEEGDD and EEEGEDD, were identified; the mass spectrum of the former sequence is more similar to that of the LMWCr fragment. Langmuir isotherm and Hill plots were used to analyze the binding constants of chromic ions to synthetic peptides similar in composition to LMWCr and apoLMWCr. The sequence pEEEEGDD can bind 4 chromic ions per peptide as apoLMWCr does, while the other sequences examined only bind two chromic ions. Studies to further elucidate the structure of LMWCr are ongoing.
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    Solution structure of the target recognition domain of zoocin A, an antibacterial enzyme, and the metal binding site of zoocin A
    (University of Alabama Libraries, 2009) Chen, Yinghua; Timkovich, Russell; University of Alabama Tuscaloosa
    A high-resolution structure has been obtained by three-dimensional NMR spectroscopy for the recombinant target recognition domain (rTRD) of zoocin A. The rTRD is a 128-residue protein responsible for targeting the cell wall of sensitive bacteria. It has a globular domain consisting of an unstructured N-terminal region, two pairs of short anti-parallel beta sheets, two sets of three-strand β-sheet and an α-helix at the c-terminal. A search for the similar fold with DALI server and SWISS-MODEL server yields no significant match, suggesting the novel folding of the rTRD. A hypothesis for the location of the binding site was proposed after observing that EDTA bound specifically to the rTRD. Chemical shifts of backbone amides of T64, G67, T70, G79, Y80 and V82 are affected by EDTA binding, and these residues are close to the poorly-structured region (residues 71-74) of the rTRD, which could provide flexibility for the rTRD to bind to its natural substrate. Additionally, the metal binding site of zoocin A has been studied by 113Cd-NMR and 15N-HSQC experiments. The two 113Cd resonances at 113.6 ppm and 107.2 ppm suggested that two nitrogen and two oxygen atoms ligate to metal center according to the correlation between the chemical shifts of Cd resonances and coordination environment of 113Cd complexes. As the metal binding site is only located at the catalytic domain (CAT) of zoocin A, two-dimensional 15N HSQC experiments for the recombinant catalytic domain (rCAT), metal-free rCAT and reconstituted Zn-rCAT provide evidence for Zn2+ as the metal cofactor. Therefore, zoocin A is also a Zn metalloprotein like its homologous proteins lysostaphy and LytM.