Synthetic Tools for the Preparation of Modified Histones
| Main Author: | |
|---|---|
| Language: | English |
| Published: |
The Ohio State University / OhioLINK
2011
|
| Subjects: | |
| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=osu1322664987 |
| id |
ndltd-OhioLink-oai-etd.ohiolink.edu-osu1322664987 |
|---|---|
| record_format |
oai_dc |
| spelling |
ndltd-OhioLink-oai-etd.ohiolink.edu-osu13226649872021-08-03T06:04:18Z Synthetic Tools for the Preparation of Modified Histones Shimko, John C. Biochemistry histone post-translational modification peptide synthesis native chemical ligation total synthesis <p>The eukaryotic genome is organized into nucleosomes consisting of 146 bp of DNA wrapped around an octamer of histone proteins, two copies each of H2A, H2B, H3, and H4. Post-translational modification (PTM) of histones perturbs nucleosome structure and dynamics thereby regulating important biological processes including transcription, replication and DNA repair. To understand these processes, we have developed synthetic tools for the preparation of homogenous samples of modified histones.</p><p>We established a novel ligation-desulfurization system for the preparation of modified histone H4 proteins enabling acetylation and phosphorylation adjacent to the C-terminus of the protein while retaining the native protein sequence. Modified H4 proteins were reconstituted into nucleosomes and nucleosome arrays. The effect of lysine 77 and 79 acetylation on nucleosome array stability was assessed. Furthermore, we demonstrated that the simultaneous incorporation of eight acetylated lysines within the LRS and dyad regions of the nucleosome does not significantly impact the structure or stability of the nucleosome. </p><p>We introduced the total synthesis of histone H3 acetylated at lysine 56 (H3-K56ac) via the chemoselective condensation of three peptide segments prepared by manual solid phase peptide synthesis (SPPS) with Boc chemistry. Non-native cysteine residues at sites of ligation were converted to native alanine residues by free-radical-desulfurization. Reconstitution and characterization of H3-K56ac semi-synthetic nucleosomes revealed that the introduced modification increases DNA site-accessibility and protein invasion of the nucleosome. We developed a reversible protection strategy for 3,4-diaminobenzoic acid (Dbz), a Fmoc compatible thioester precursor, which allows for the use of highly activating conditions and acetyl capping during Fmoc peptide synthesis. Dbz protection maximizes product yield while minimizing the formation of deletion products. Further, we demonstrated the novel site-specific derivitization of the unsubstituted Dbz amine with biophysical probes including biotin and fluorophores.</p><p>Finally, we adapted our initial total synthesis strategy for modified histone H3 to allow for the automated synthesis of a library of ~45 residue-modified peptide segments in high yield and purity by Fmoc chemistry. The combination of automated peptide synthesis protocols with sequential NCL allows for a widely accessible strategy for the combinatorial preparation of differentially-modified histone proteins suitable for biophysical characterization.</p> 2011-12-19 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1322664987 http://rave.ohiolink.edu/etdc/view?acc_num=osu1322664987 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. |
| collection |
NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Biochemistry histone post-translational modification peptide synthesis native chemical ligation total synthesis |
| spellingShingle |
Biochemistry histone post-translational modification peptide synthesis native chemical ligation total synthesis Shimko, John C. Synthetic Tools for the Preparation of Modified Histones |
| author |
Shimko, John C. |
| author_facet |
Shimko, John C. |
| author_sort |
Shimko, John C. |
| title |
Synthetic Tools for the Preparation of Modified Histones |
| title_short |
Synthetic Tools for the Preparation of Modified Histones |
| title_full |
Synthetic Tools for the Preparation of Modified Histones |
| title_fullStr |
Synthetic Tools for the Preparation of Modified Histones |
| title_full_unstemmed |
Synthetic Tools for the Preparation of Modified Histones |
| title_sort |
synthetic tools for the preparation of modified histones |
| publisher |
The Ohio State University / OhioLINK |
| publishDate |
2011 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1322664987 |
| work_keys_str_mv |
AT shimkojohnc synthetictoolsforthepreparationofmodifiedhistones |
| _version_ |
1719430374003572736 |