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You searched for subject:(arginine affinity). Showing records 1 – 2 of 2 total matches.

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Miami University

1. New, Christopher Paul. Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport.

Degree: PhD, Cell, Molecular and Structural Biology (CMSB), 2020, Miami University

The chloroplast Twin Arginine Translocase (cpTAT) system transports fully folded proteins across the thylakoid membrane in plant cells using only energy derived from the proton motive force (PMF). Three membrane bound component proteins: cpTatC, Hcf106, and Tha4 function together in a transient manner to accomplish transport. However, clear mechanistic details of this process remain elusive such as how cpTAT utilizes energy stored in the PMF or how the individual component proteins interact during each step of transport. In addition, prior structural characterization of (cp)TAT proteins used truncated versions of the components. This dissertation describes work to develop methods to purify full-length Hcf106 for biophysical characterization. Additionally, this dissertation details the work to determine the function of a membrane embedded glutamate in the Tha4 transmembrane helix (TMH).A series of purification trials were carried out to isolate Hcf106 fused to maltose binding protein (MBP) by the recognition sequence of tobacco etch virus protease (TEVp). Fusion protein and protease were expressed in and purified from E. coli using affinity chromatography. Multiple parameters and additives were tested during optimization of TEVp proteolysis reactions with MBP-Hcf106. TEVp and free MBP were separated from un-cleaved MBP-Hcf106 and free Hcf106 by affinity and size exclusion chromatography. Although TEVp and free MBP were removed after an optimized proteolysis reaction, free Hcf106 showed its recalcitrant nature through resistance of separation from un-cleaved MBP-Hcf106 by size exclusion chromatography in several detergent and buffer conditions.To better understand the role of the membrane embedded Tha4 glutamate 10 (E10), Tha4 variants with glutamate to alanine (E10A) or glutamate to aspartate (E10D) substitutions were used to complement loss of cpTAT function in thylakoid membranes. Sequential glutamate substitutions in the TMH of Tha4 variant E10A were unable to restore transport while aspartate substitutions were mildly able to complement loss of function. Furthermore, organization between three structural regions in Tha4 E10/A/D variants was determined by disulfide crosslinking during various transport conditions. Tha4 E10/A/D variant oligomer formation was enhanced in the presence of functional precursor with and without PMF present. An increase in TMH hydrophobicity by alanine substitution was shown to increase Tha4 stability in isolated thylakoid membranes and to promote tighter packing interactions between adjacent Tha4 monomers. The interaction data was then used to develop a model of how Tha4 E10/A/D variant tetramers pack and reorganize in the presence of precursor. Advisors/Committee Members: Dabney-Smith, Carole (Advisor), Page, Rick (Committee Chair).

Subjects/Keywords: Biochemistry; Cellular Biology; Plant Biology; Molecular Biology; chloroplast twin arginine transport; protein transport; cpTAT; TAT; Tha4; Hcf106; protein purification; maltose binding protein affinity chromatography; oligomer formation; complementation; transmembrane domain hydrophobicity

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

New, C. P. (2020). Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport. (Doctoral Dissertation). Miami University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538

Chicago Manual of Style (16th Edition):

New, Christopher Paul. “Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport.” 2020. Doctoral Dissertation, Miami University. Accessed January 18, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538.

MLA Handbook (7th Edition):

New, Christopher Paul. “Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport.” 2020. Web. 18 Jan 2021.

Vancouver:

New CP. Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport. [Internet] [Doctoral dissertation]. Miami University; 2020. [cited 2021 Jan 18]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538.

Council of Science Editors:

New CP. Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport. [Doctoral Dissertation]. Miami University; 2020. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538


University of St. Andrews

2. Carr, Sharon. Adenovirus and its interaction with host cell proteins .

Degree: 2007, University of St. Andrews

The E1B55k gene in adenovirus type 5 was studied by generating recombinant viruses in which the E1B55k gene was N-terminally tagged with either a cyan fluorescent protein (CFP) tag or a tandem affinity purification (TAP) tag. By infecting cells with the recombinant adenovirus expressing CFP tagged E1B55k, localisation of the E1B55k protein during infection could be studied. Generation of the recombinant viruses expressing TAP tagged E1B55k would allow purification of the E1B55k protein and any cellular or viral proteins that interacted with E1B55k. By infecting different cell types and harvesting at set time points during infection, the proteins interacting with E1B55k at different time points during infection could be identified. The E1B55k protein is modified by small ubiquitin modifying protein 1 (SUMO-1) at lysine residue 104 within the SUMO consensus sequence, and sumoylation can be abolished by mutating the lysine residue to an arginine (Endter et al, 2001). Therefore in order to study the effect of SUMOylation of E1B55k, on adenovirus infection, CFP and TAP tagged recombinant viruses in which the E1B55k protein was mutated to contain an unmodificable arginine at postion 104 were also generated. Stable cell lines expressing TAP tagged wild type E1B55k, and E1B55k in which lysine residue 104 had been mutated to an arginine were also generated as an alternative method to study the interacting proteins of E1B55k. Four different isoforms of SUMO have been identified, named SUMO1-4. Stable cell lines expressing SUMO-2 were infected with wild type adenovirus type 5 and harvested at set time points in order to determine if any adenoviral proteins were modified by SUMO-2. Advisors/Committee Members: Botting, Catherine (advisor), Hay, Ronald T (advisor).

Subjects/Keywords: Adenovirus; E1B55k; SUMO; Recombinant; Tandem affinity purification; TAP; CFP; Cyan fluorescent protein; Lysine to arginine mutation; SUMO consensus sequence

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Carr, S. (2007). Adenovirus and its interaction with host cell proteins . (Thesis). University of St. Andrews. Retrieved from http://hdl.handle.net/10023/219

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Carr, Sharon. “Adenovirus and its interaction with host cell proteins .” 2007. Thesis, University of St. Andrews. Accessed January 18, 2021. http://hdl.handle.net/10023/219.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Carr, Sharon. “Adenovirus and its interaction with host cell proteins .” 2007. Web. 18 Jan 2021.

Vancouver:

Carr S. Adenovirus and its interaction with host cell proteins . [Internet] [Thesis]. University of St. Andrews; 2007. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/10023/219.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Carr S. Adenovirus and its interaction with host cell proteins . [Thesis]. University of St. Andrews; 2007. Available from: http://hdl.handle.net/10023/219

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

.