Stability and folding studies of ribonuclease T1 /
The folding pathway of ribonuclease T1 was investigated using circular permutation to reorder the secondary structural units within the protein. This was accomplished using a PCR protocol to systematically investigate the importance of all the solvent exposed loops and turns by inserting new termin...
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| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
1996.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=739667801&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The folding pathway of ribonuclease T1 was investigated using circular permutation to reorder the secondary structural units within the protein. This was accomplished using a PCR protocol to systematically investigate the importance of all the solvent exposed loops and turns by inserting new termini within them. Four of the circularly permuted proteins cp35S1, cp49D1, cp7OG1, and cp96S1 correctly folded and exhibited similar structure and catalytic activity to the wild-type protein. Within the tertiary structure of RNase T1, there is a single turn, between residues 82 and 85, which may nucleate the folding of RNase T1. The importance of this []-turn was assessed by circularly permuting the primary sequence, creating new termini within this loop, and inserting a pair of cysteines at the new termini which could be selectively reduced to demonstrate the necessity of this turn for proper protein folding and thermodynamic stability. A library of peptide linkers predisposed to form a turn was constructed to assess the importance of the peptide sequence used to connect the original termini of the circularly permuted proteins. A total of 12 sequences were analyzed which displayed good activity on a calorimetric plate assay. An inspection of crystal structures with peptide sequences which matched the linker sequences was conducted and used to explain differences in the stabilities of the linker proteins. Specific hydrogen-bonding patterns which stabilized the turns present in the crystal structures might also stabilize the geometry of the linker. Stabilized linkers were hypothesized to also increase the thermodynamic stability of the protein. The role of the 6-103 disulfide bond was examined by replacing the cysteines with residue pairs which mimicked the characteristics of the cysteines. Four replacements were made which approximated the characteristics of the disulfide bond which were thought to be important. The cysteines were replaced with a pair of serines, a histidine and an aspartate, a methionine and an alanine, and a pair of alanines. None of the proteins were isolated in which the 6-103 disulfide bond was absent. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xii, 149 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 143-148. |