Kinetic and thermodynamic analysis of Bradyrhizobium japonicum PutA-membrane associations.
In Escherichia coli, proline induces tight membrane binding of the PutA flavoenzyme and transforms PutA from a transcriptional repressor to a membrane-associated proline catabolic enzyme. In other gram-negative bacteria such as Bradyrhizobium japonicum, PutA lacks DNA binding activity and functions only as a proline catabolic enzyme. Here, we characterize the membrane binding properties of PutA from B. japonicum (BjPutA) to address whether proline regulates BjPutA-lipid binding similar to Escherichia coli PutA (EcPutA). Surface plasmon resonance (SPR) kinetic measurements of BjPutA-lipid binding show BjPutA forms a complex with lipids in the absence and presence of proline with similar dissociation constant (K(D)) values of 2.5 and 1.7nM, respectively. SPR experiments using differently charged lipid bilayers indicate BjPutA selectively binds negatively charged lipids, which contrasts with the charge independent membrane binding of EcPutA. Analysis of BjPutA-lipid binding by isothermal titration calorimetry at 25 degrees C revealed an endothermic binding reaction that is entropically driven. This work shows that BjPutA-membrane associations vary significantly from EcPutA.
Zhang
Weimin
W
Department of Biochemistry, Redox Biology Center, University of Nebraska, Lincoln, NE 68588, USA.
Krishnan
Navasona
N
Becker
Donald F
DF
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020369:Bradyrhizobium / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008567:Membranes, Artificial; D010084:Oxidation-Reduction; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D008567:Membranes, Artificial; C080683:PutA protein, Bacteria; D011392:Proline