Exploring the proline-dependent conformational change in the multifunctional PutA flavoprotein by tryptophan fluorescence spectroscopy.
The multifunctional PutA flavoprotein regulates proline utilization in Escherichia coli by switching from a cytosolic DNA-binding protein to a membrane-bound enzyme with proline dehydrogenase (PRODH) and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities. The transformation of PutA from a transcriptional repressor of the proline utilization (put) regulon to a peripheral membrane associated enzyme is mediated by a proline-dependent conformational change. Previously, limited proteolysis of PutA indicated that the conformational change involves a flexible domain of unknown function (residues 141-262) which is nearby the FAD-binding and PRODH active sites (residues 263-610). Here, we extend our understanding of the proline-dependent conformational change in PutA by investigating the intrinsic Trp fluorescence spectroscopic properties of a truncated PutA protein which contains residues 86-601 (PutA86-601) and only four Trp residues. The addition of proline to wild-type PutA86-601 decreases Trp fluorescence by 36%, indicating a substantial conformational change. An apparent rate constant of 0.59 +/- 0.06 s(-)(1) was determined for the fluorescence change by stopped-flow fluorescence measurements. The limiting rate constant for proline reduction of the FAD cofactor in PutA is 133 +/- 6 s(-)(1), demonstrating that FAD reduction precedes the conformational transition observed by Trp fluorescence. The nonreducing ligand l-tetrahydro-2-furoic acid mimics the decrease in Trp fluorescence induced by proline, indicating that both FAD reduction and ligand binding contribute to the observed conformational change in PutA86-601. W194 and W211, which are located in the flexible domain, were replaced by Phe in the PutA86-601 mutants W194F, W211F, and W194F/W211F to determine which residue is involved in the observed fluorescence change. Analysis of the PutA86-601 mutants indicated that W211 is the primary molecular marker of the conformational change caused by proline. Altogether, this work shows that the switching of PutA from a transcriptional repressor to a membrane-bound protein involves W211 in a flexible domain near the PRODH active site and occurs on a time scale that is >10-fold slower than the turnover number of PutA.
Zhu
Weidong
W
Department of Biochemistry, Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588, USA.
Becker
Donald F
DF
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000378:metabolism; D011392:Proline; D011394:Proline Oxidase / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D001426:Bacterial Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria; D014364:Tryptophan; D011392:Proline; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011394:Proline Oxidase