Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex

Ferredoxin-NADP+ reductase (FNR) was previously inferred to bind to the cytochrome b6f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b(6)f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b(6)f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b(6)f interaction is explained by FNR interaction with micelles of the undecyl 6-D maltoside (UDM) detergent micelles used to purify b(6)f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP(+)-binding domains of FNR caused by detergent. The enthalpy, Delta H-o, and the entropy, Delta S-o, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b(6)f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins. SIGNIFICANCE This study concerns structure properties and membrane-protein interactions of the cytochrome b(6)f complex for which a 2.5 A crystal structure has been obtained. Regarding electron transfer between FNR and b(6)f complex, a question is whether FNR, an electron donor to the cytochrome b(6)f complex, interacts with the complex to an extent that both proteins should be considered part of a "super-complex". This inference had been based on a tendency for co purification and electron exchange. Here, isothermal titration calorimetry was used to examine the existence of a significant interaction between FNR and the b(6)f complex. Such an interaction was not found. Co-purification of FNR with the b(6)f complex is associated with a significant affinity of FNR for undecyl 6-D maltoside detergent.