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You searched for +publisher:"Miami University" +contributor:("Page, Rick"). Showing records 1 – 3 of 3 total matches.

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Miami University

1. Moseng, Mitchell A. THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS.

Degree: PhD, Chemistry, 2019, Miami University

URL: http://rave.ohiolink.edu/etdc/view?acc_num=miami1563537216760463 

HSPA9, the gene coding for the mitochondrial chaperone mortalin, is involved in various cellular roles such as mitochondrial protein import, folding, degradation, Fe-S cluster biogenesis, mitochondrial homeostasis, and regulation of the anti-apoptotic protein p53. Due to the crucial role in cell survivability mortalin is also an aim for drug design with a strong emphasis on anticancer treatments. Mutations in the HSPA9 gene, particularly within the region coding for the nucleotide-binding domain (NBD), cause the autosomal disorder identified as EVEN-PLUS syndrome. The resulting mutants R126W and Y128C are located on the exterior of the mortalin-NBD near the interface of the interdomain linker (IDL). We used differential scanning fluorimetry (DSF), biolayer interferometry, X-ray crystallography, ATP hydrolysis assays, and Rosetta docking simulations to study the structural and functional consequences of the EVEN-PLUS syndrome-associated R126W and Y128C mutations within the mortalin-NBD. In order to determine a potential mortalin inhibitor we screened adenosine-5’-diphosphate (ADP) analogs by isothermal titration calorimetry (ITC) and inhibition assays. As part of our initial efforts to determine a promising covalent inhibitor of mortalin we crystallized human mortalin nucleotide binding domain (NBD) with N6-propargyl ADP. The acquired structure highlighted the ability of the nucleotide binding pocket to accommodate modified ADP compounds and reveals two possible sites for modification on nucleotides to increase specificity for mortalin. A library of ADP analogues containing modifications at either the 2C or N6 positions of adenosine were screened against mortalin-NBD to determine binding affinities. These results of the surface mutation studies indicate that the R126W and Y128C mutations have a far-reaching effect and disrupt ATP hydrolysis, interdomain linker binding, and thermostability. The structural differences observed provide insight into how the conformations of mortalin vary from other heat shock protein 70 (Hsp70) homologs. Combined, our biophysical and structural studies contribute to the understanding of the molecular basis for how disease-associated mortalin mutations affect mortalin functionality and the pathogenesis of EVEN-PLUS syndrome. The results of competitive inhibition and binding assays of the analogs demonstrate that modifications at the 2C or N6 positions have potential to bind and inhibit mortalin uniquely compared to other Hsp70 homologs. In particular, this data indicates that modifications at the 2C position confer the greatest selectivity in binding and inhibition of mortalin-NBD compared to the cytosolic homologs, Hsc70 and Hsp70. Advisors/Committee Members: Page, Rick (Advisor).

Subjects/Keywords: Biochemistry; Mortalin

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

APA (6th Edition):

Moseng, M. A. (2019). THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS. (Doctoral Dissertation). Miami University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=miami1563537216760463

Chicago Manual of Style (16th Edition):

Moseng, Mitchell A. “THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS.” 2019. Doctoral Dissertation, Miami University. Accessed January 18, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=miami1563537216760463.

MLA Handbook (7th Edition):

Moseng, Mitchell A. “THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS.” 2019. Web. 18 Jan 2021.

Vancouver:

Moseng MA. THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS. [Internet] [Doctoral dissertation]. Miami University; 2019. [cited 2021 Jan 18]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1563537216760463.

Council of Science Editors:

Moseng MA. THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS. [Doctoral Dissertation]. Miami University; 2019. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1563537216760463


Miami University

2. 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

URL: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538 

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

3. Habtemichael, Aman Gebreyohannes. Insights into the Chloroplast Tat Mechanism of Transport.

Degree: MS, Chemistry, 2017, Miami University

URL: http://rave.ohiolink.edu/etdc/view?acc_num=miami1500654052065743 

The chloroplast Twin arginine transport (cpTat) system transports folded proteins across the thylakoids in chloroplast using proton motive force as the only source of energy. The cpTat is composed of three components; Tha4, Hcf106, and cpTatC. Hcf106 and cpTatC form a receptor complex where the precursor binds. Tha4 is found as separate homo-oligomers that assemble the receptor-bound substrate to complete the translocation. Though the overall model for the transport cycle is established, detailed sequential events remain to be elucidated. This thesis mainly probes conformational change of Hcf106 upon substrate binding using cysteine accessibility technique and interaction points between cpTatC first stromal domain (S1) and precursor mature domain using disulfide crosslinking to understand their direct contribution to the actual translocation event. We detected a more buried Hcf106 amphipathic helix (APH) after precursor binds. Besides, interactions were detected between cpTatC S1 and precursor mature domain. Altogether, our data supports a model that depicts Hcf106 APH membrane weakening and cpTatC insertase activity to promote the translocation of the precursor. Advisors/Committee Members: Dabney-Smith, Carole (Advisor), Page, Rick (Committee Chair).

Subjects/Keywords: Biochemistry; Thylakoid; protein transport; chloroplast twin arginine transport

…time at Miami University. You have inspired me to become an independent researcher and what a… 

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

APA (6th Edition):

Habtemichael, A. G. (2017). Insights into the Chloroplast Tat Mechanism of Transport. (Masters Thesis). Miami University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=miami1500654052065743

Chicago Manual of Style (16th Edition):

Habtemichael, Aman Gebreyohannes. “Insights into the Chloroplast Tat Mechanism of Transport.” 2017. Masters Thesis, Miami University. Accessed January 18, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=miami1500654052065743.

MLA Handbook (7th Edition):

Habtemichael, Aman Gebreyohannes. “Insights into the Chloroplast Tat Mechanism of Transport.” 2017. Web. 18 Jan 2021.

Vancouver:

Habtemichael AG. Insights into the Chloroplast Tat Mechanism of Transport. [Internet] [Masters thesis]. Miami University; 2017. [cited 2021 Jan 18]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1500654052065743.

Council of Science Editors:

Habtemichael AG. Insights into the Chloroplast Tat Mechanism of Transport. [Masters Thesis]. Miami University; 2017. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1500654052065743

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