Miller, Stefan and Schuler, Benjamin and Seckler, Robert (1998) Phage P22 tailspike protein: Removal of head-binding domain unmasks effects of folding mutations on native-state thermal stability. PROTEIN SCIENCE, 7 (10). pp. 2223-2232. ISSN 0961-8368,
Full text not available from this repository. (Request a copy)Abstract
A shortened, recombinant protein comprising residues 109-666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified from Escherichia coli and crystallized. Like the full-length tailspike, the protein lacking the aminoterminal head-binding domain is an SDS-resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature-sensitive-folding (tsf) mutations and the four known global second-site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full-length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full-length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant-induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild-type protein in the presence of 2% SDS at 71 degrees C occurs at a rate of 9.2 x 10(-4) s(-1). It reflects the second kinetic phase of unfolding of the full-length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10-fold. Two of the su mutations retard thermal unfolding up to 5-fold, while the remaining two mutations accelerate unfolding up to 5-fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | SODIUM-DODECYL-SULFATE; TAIL SPIKE ENDORHAMNOSIDASE; INCLUSION-BODY FORMATION; CRYSTAL-STRUCTURE; IN-VITRO; GLOBAL SUPPRESSORS; PATHWAY; INTERMEDIATE; INVITRO; SITES; bacteriophage tail protein; parallel beta-helix; protein folding intermediates; protein stability; temperature-sensitive mutants |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 18 Apr 2023 13:56 |
| Last Modified: | 18 Apr 2023 13:56 |
| URI: | https://pred.uni-regensburg.de/id/eprint/49481 |
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