The development of eclosion hormone as a model protein and the use of matrix-assisted laser desorption ionization mass spectrometry to study protein folding and stability /
Manduca sexta Eclosion Hormone (EHQ) and its two analogs
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| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
1995.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=742536471&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Manduca sexta Eclosion Hormone (EHQ) and its two analogs EHN and EHS were synthesized by Fmoc solid phase methodology. Using matrixassisted laser desorption ionization mass spectrometry (MALDI-MS), a purification scheme was developed. The purified unfolded proteins were characterized via amino acid analysis and N-terminal sequence analysis. The folding of the EH analogs were monitored with MALDI-MS. EH and their analogs were tested for biological activity with an in vivo bioassay using pharate adult Heliothis virescens. The successful synthesis and folding of the EH analogs resulted in biologically active proteins. Two other small proteins with three disulfide bonds, bovine insulin and bovine pancreatic trypsin inhibitor (BPTI), were used to develop the methodology of utilizing MALDIMS to monitor Cys reduction and alkylation conditions, to determine the &sulfide pairing pattern in the folding intermediate, to monitor protein refolding, and to estimate the protein stability. Reduced BPTI was completely oxidized and refolded in the glutathione redox buffer in the presence of a denaturant, guanidine hydrochloride (Gdn.HCI); while in the absence of the denaturant, the refolding yield was about only 70%. Protein stability estimation using MALDI-MS suggested that EHS is less stable than BPTI. The estimated relative stability of BPTI and EHS was in a good agreement with the predicted values from transfer free energy (TFE) model. |
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| Item Description: | Vita. "Major Subject: Chemical Engineering". |
| Physical Description: | x, 99 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |