Conformational stability of ribonuclease Sa from Streptomyces aureofaciens /
A chemically synthesized gene for ribonuclease Sa, RNase Sa,
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| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
1997.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=736583741&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | A chemically synthesized gene for ribonuclease Sa, RNase Sa, from Streptomyces aureofaciens BM-K has been cloned and expressed in Escherichia coli. A simple two column purification scheme has been developed for the purification of - 60 mg of recombinant protein per liter of liquid culture. The recombinant protein expressed in E. coli is identical to the protein produced by the Actinomycete, S. aureofaciens. Thermal denaturation has been used at several different pH values to investigate the conformational stability of the protein. From these data the free energy of unfolding, AGu (5.3 kcal mol-1 at 25 'C, 30 mM MOPS pH 7.0), enthalpy of unfolding, AH (90.1 kcal mol-1), the melting temperature, Tm (48.6 'C), and heat capacity change associated with unfolding, ACp (1.45 kcal mol-1 K'), have been measured. The pH dependence and salt dependence of the conformational stability of RNase Sa have also been studied. Seven hydrogen bonding mutants in RNase Sa have been made to estimate the contribution of hydrogen bonding interactions to the conformational stability of RNase Sa., From thermal denaturation studies of these mutants, hydrogen bonding is estimated to contribute on average - 3 kcal mol-1 per hydrogen bond to the stability of the native state of RNase Sa. This estimate is slightly higher than that estimated from 52 hydrogen bonding mutants from six different proteins analyzed previously (Myers & Pace, 1996). The large variation in the measured A(AG) values for hydrogen bonding interactions have been examined for several similar hydrogen bonding interactions in the RNases Sa, Tl, and Ba. We find no correlations for the bond length and bond angles to hydrogen bonding strength. A single tryptophan residue has been added to the wild-type sequence of RNase Sa to provide an additional spectroscopic probe to monitor protein unfolding. The Tyr52-4Trp RNase Sa mutant shows a 4,, of 311 mn in the folded state and shows a seven fold decrease in fluorescence intensity upon unfolding in 9M urea. This tryptophan is the second most blue shifted tryptophan found in proteins. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xv, 151 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 134-150. |