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Redesigned purification yields a fully functional PutA protein dimer from Escherichia coli.
The Journal of Biological Chemistry
|June 25, 1992
Summary
The PutA protein in E. coli and Salmonella typhimurium is crucial for proline utilization, acting as both a repressor and dehydrogenase. This study details its purification and characterization, confirming its dual functions.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Proline utilization in E. coli and S. typhimurium is regulated by the putA gene.
- Genetic studies suggest the PutA protein has dual roles: proline utilization repressor and dehydrogenase.
Purpose of the Study:
- To purify and characterize the PutA protein from E. coli.
- To confirm the physical and biological properties of the PutA protein.
Main Methods:
- Redesigned protein purification procedure.
- Electrophoresis (denaturing and nondenaturing) for homogeneity assessment.
- In vitro electrophoretic band-shift assay for DNA binding.
- Reconstitution into inverted membrane vesicles.
- Hydrodynamic analysis (Stokes radius, Svedberg coefficient).
Main Results:
- Homogeneous PutA protein was purified, with N-terminal sequence matching DNA predictions.
- Proline and delta 1-pyrroline-5-carboxylate dehydrogenase activities were copurified.
- Purified PutA protein demonstrated binding to put DNA in vitro.
- Reconstitution into membrane vesicles restored proline dehydrogenase activity.
- Hydrodynamic data indicated a dimeric, irregularly shaped protein (293 kDa).
Conclusions:
- The PutA protein possesses both DNA-binding repressor and dehydrogenase activities.
- Purified PutA protein functions in proline metabolism and transport.
- The study provides detailed biophysical characterization of the PutA protein.