Caged thiophosphorylated peptides and proteins /
An SH2 binding peptide containing the YEEI motif, and a PKA catalytic subunit were chosen to explore the caging of signaling polypeptides at crucial tyrosine or threonine phosphorylation sites. The interaction between phosphorylated tyrosine residue and SH2 domain (pTyr-SH2) mediates countless prot...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2001.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=726103621&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | An SH2 binding peptide containing the YEEI motif, and a PKA catalytic subunit were chosen to explore the caging of signaling polypeptides at crucial tyrosine or threonine phosphorylation sites. The interaction between phosphorylated tyrosine residue and SH2 domain (pTyr-SH2) mediates countless protein-protein interactions. Phosphorylation can be mimicked by thiophosphorylation. To use light to control the pTyr-SH2 association, a thiophosphate group was introduced into peptide EPQYEEIPILG at the tyrosine residue by kinase Hck and ATPγS. The peptide was then caged on the thiophosphate group with 2-nitrobenzyl bromide (NBB) or 4-hydroxyphenacyl bromide (HPB). Photolysis was performed by irradiation at 312 nm. EPQYpsEEIPILG was released from EPQYps(NB)EEIPILG in 50-60% yield. The t₁/₂ values were 5.5 min at pH5.8 and 8.0 min at pH7.3, which correspond to quantum yields (øp) of 0.37 and 0.25. EPQYpsEEIPILG was produced from EPQYps(HP)EEIPILG in a yield of 50-70% at both pH 5.8 and pH 7.3. The t₁/₂ values were 0.55 min at pH 5.8 and 0.52 min at pH 7.3, which correspond to (øp) values of 0.65 and 0.56. When the binding ability of caged³⁵S-labeled peptides to immobilized SH2 was examined, only 1 to 4 % of the radioactivity was bound, which largely represents non-specific binding. Upon photolysis, in both cases, EPQYps(NB)EEIPILG and EPQYps(HP)EEIPILG, the binding ability of the peptide was restored. Phosphothreonine 197 is crucial for the activity of the PKA catalytic subunit. Nonphosphorylated, inactive protein H₆-C199A/C343A was obtained by expression in the presence of H89, a specific inhibitor of PKA, to prevent autophosphorylation. A thiophosphate group was introduced at Thr 197 by the kinase PDK1 and ATP(γ)S, resulting in an active protein. The thiophosphorylated protein was purified by TNB-thiol agarose. Caging of the protein with HPB led to inactivation with a 20-fold reduction in activity. Upon photolysis, the protein was released with a t₁/₂ of 1.5 min and øp of 0.2. Regeneration of the thiophosphate group was determined by the increase in coupling ability to TNB-thiol gel, an activated disulfide-containing resin. Concomitant with the increased binding was a 15-18 fold increase in catalytic activity. |
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| Item Description: | Vita. "Major Subject: Genetics". |
| Physical Description: | xi, 153 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 135-152). |