Development and Application of Chemical and Structural Biology Approaches to Probe Protein Function
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| Language: | English |
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The Ohio State University / OhioLINK
2011
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=osu1306439016 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu1306439016 |
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oai_dc |
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NDLTD |
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English |
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Biochemistry Biophysics membrane protein crystallography Rh protein Rhesus channel CO2 ammonium carbonic anhydrase pyrrolysine nonnatural amino acid genetic code expansion intein click chemistry ubiquitin ubiquitination native chemical ligation semisynthesis |
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Biochemistry Biophysics membrane protein crystallography Rh protein Rhesus channel CO2 ammonium carbonic anhydrase pyrrolysine nonnatural amino acid genetic code expansion intein click chemistry ubiquitin ubiquitination native chemical ligation semisynthesis Li, Xin Development and Application of Chemical and Structural Biology Approaches to Probe Protein Function |
| author |
Li, Xin |
| author_facet |
Li, Xin |
| author_sort |
Li, Xin |
| title |
Development and Application of Chemical and Structural Biology Approaches to Probe Protein Function |
| title_short |
Development and Application of Chemical and Structural Biology Approaches to Probe Protein Function |
| title_full |
Development and Application of Chemical and Structural Biology Approaches to Probe Protein Function |
| title_fullStr |
Development and Application of Chemical and Structural Biology Approaches to Probe Protein Function |
| title_full_unstemmed |
Development and Application of Chemical and Structural Biology Approaches to Probe Protein Function |
| title_sort |
development and application of chemical and structural biology approaches to probe protein function |
| publisher |
The Ohio State University / OhioLINK |
| publishDate |
2011 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1306439016 |
| work_keys_str_mv |
AT lixin developmentandapplicationofchemicalandstructuralbiologyapproachestoprobeproteinfunction |
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1719429990845513728 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu13064390162021-08-03T06:02:41Z Development and Application of Chemical and Structural Biology Approaches to Probe Protein Function Li, Xin Biochemistry Biophysics membrane protein crystallography Rh protein Rhesus channel CO2 ammonium carbonic anhydrase pyrrolysine nonnatural amino acid genetic code expansion intein click chemistry ubiquitin ubiquitination native chemical ligation semisynthesis <p>My research in The Ohio State University is focused on the development and application of structural and chemical biology approaches to probe protein function.</p><p>In the first chapter, the challenges and advances in the field of membrane protein crystallography are discussed. X-ray crystallography is so far the most powerful technique to characterize the structural details of proteins. However its application on the membrane proteins was lagged due to the special properties of this class of proteins. New methodologies have been developed in recent years to facilitate membrane protein structure determination by adapting existing methods for soluble proteins. </p><p>In the second chapter, the structure of an integral membrane protein, Rh protein, was determined by X-ray crystallography and its functional role and molecular mechanism were deduced and discussed from the structural data. Rh proteins, best known for their role as a human blood factor and antigen, are membrane channels that transport gas molecules, but it’s in debating which, ammonia or CO<sub>2</sub>, is the physiological substrate of Rh protein. A structural biology approach is taken here to address this substrate issue by determining the first structure of an Rh family member, the Rh protein from <i>Nitrosomonas europaea</i>. This Rh protein exhibits a number of similarities to bacterial and archael ammonium transporters, including a trimeric state, a twin-His-centered central channel, and a Phe residue that blocks the channel. However, there are some significant differences, including an additional cytoplasmic C-terminal helix, an increased number of internal prolines along the transmembrane helices, as well as a specific set of residues linking the C-terminal helix to Phe blockage. This linkage suggests a possible mechanism in which binding of a partner protein to the C-terminus could regulate channel opening. Another difference observed in the Rh structure is the absence of the extracellular-cation binding site thought to recruit ammonium. Instead, a CO<sub>2</sub>-binding site near the intracellular exit of the channel is identified. The implications of these findings on the functional role of the human Rh antigens are discussed. In the third chapter, the function and the structure of <i>N. europaea</i> α-carbonic anhydrase, a potential Rh protein partner protein, were characterized in detail. Carbonic anhydrases are a group of Zn containing enzymes catalyzing the interconversion between CO<sub>2</sub> and bicarbonate. The physiological functions of carbonic anhydrases in prokaryotes are mostly unclear. The cloning, purification, biochemical characterization, and 1.45 Angstrom crystal structure of N. europaea α-carbonic anhydrase are reported here. This enzyme has a significantly reduced activity. Subsequent crystallographic studies reveal an unidentified ligand bound to the active center. The origin of the ligand and the potential application using it as carbonic anhydrase targeting inhibitor are discussed.</p><p>In the fourth chapter, a new chemical biology approach to site-specifically label proteins is developed. This technology is based on the discovery of the 22nd genetically encoded amino acid, pyrrolysine, in some archaea methanogens. Pyrrolysine is incorporated into proteins in response to the UAG codon in these organisms, which have specialized tRNA<sub>Pyl</sub> and pyrrolysyl-tRNA<sub>Pyl</sub> synthetase. Introducing these two factors into <i>E. coli</i> enables <i>E. coli</i> to read the UAG codon as a sense codon and incorporate pyrrolysine or a pyrrolysine analog supplied in the growth medium. A highly convenient UAG codon readthrough assay is described that facilitates testing the newly synthesized analogs. Using this assay, two pyrrolysine analogs containing an acetylene group suitable for attaching functional tags (fluorescent dyes, biotin) by click chemistry could be identified. Using this pyrrolysine-base labeling technology, fluorescently labeled calmodulin could be prepared, and its Ca<sup>2+</sup>-binding peptide mediated conformational changes monitored by either Forster resonance energy transfer (FRET) or traditional fluorescence techniques. </p><p>In the fifth chapter, a semisynthetic method to generate ubiquitinated proteins for the structural and functional studies is developed. Ubiquitination is one of the most important post-translational protein modifications. Unfortunately, the biochemical and structural characterization of ubiquitinated proteins has been significantly hindered by the difficulty in their preparation. This challenge is addressed here by developing a facile method to prepare site-specifically ubiquitinated. This method utilizes the pyrrolysine incorporation system to genetically encode a cysteine-containing pyrrolysine analog into the protein targeted for ubiquitination. The resulting modified calmodulin is then allowed to react via native chemical ligation to a ubiquitin thioester generated from an intein-fusion. The significance of this approach is that it provides a potentially general strategy to prepare many different ubiquitinated proteins. This technology has been successfully exploited to generate a monoubiquitined protein and an unconventional diubiquitin (diUb). Biochemical and crystallographic studies on these semisynthetic ubiquitinated proteins have led to new insights of the functional and structural properties of ubiquitination.</p> 2011-07-25 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1306439016 http://rave.ohiolink.edu/etdc/view?acc_num=osu1306439016 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |