Abstract
A structural analysis of the surface areas of cytochrome c(6), responsible for the transient interaction with photosystem I, was performed by NMR transverse relaxation-optimized spectroscopy. The hemeprotein was titrated by adding increasing amounts of the chlorophyllic photosystem, and the NMR spectra of the free and bound protein were analyzed in a comparative way. The NMR signals of cytochrome c(6) residues located at the hydrophobic and electrostatic patches, which both surround the heme cleft, were specifically modified by binding. The backbones of internal residues close to the hydrophobic patch of cytochrome c(6) were also affected, a fact that is ascribed to the conformational changes taking place inside the hemeprotein when interacting with photosystem I. To the best of our knowledge, this is the first structural analysis by NMR spectroscopy of a transient complex between soluble and membrane proteins.
Highlights
NMR spectroscopy makes it possible to observe the signals of particular atoms in the free and bound states of any molecule [1] and has become an essential tool for the analysis of protein-protein interactions [2]
Conventional solution NMR spectroscopy is usually limited to complexes with a molecular mass lower than 50 kDa because of their quick tumbling, and it relies on the average of orientationdependent dipolar interactions and chemical shifts
We investigated the residues of cytochrome c6 involved in the interaction with photosystem I (PSI) by using Transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy
Summary
NMR spectroscopy makes it possible to observe the signals of particular atoms in the free and bound states of any molecule [1] and has become an essential tool for the analysis of protein-protein interactions [2]. The analysis of line-broadening perturbations of the PSI-bound hemeprotein with respect to free cytochrome c6 was performed in SPARKY 3.2 The sequential assignment of cytochrome c6 has been reported previously [17].
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