Surface interactions in the complex between cytochrome f and the E43Q/D44N and E59K/E60Q plastocyanin double mutants as determined by 1H-NMR chemical shift analysis |
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| Authors: | Anders Bergkvist Mikael Ejdebck Marcellus Ubbink and B Gran Karlsson |
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| Institution: | 1.Biochemistry and Biophysics, Department of Chemistry, Göteborg University, SE-405 30 Göteborg, Sweden ;2.Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands ;3.Department of Molecular Biotechnology, Chalmers University of Technology, SE-405 30 Göteborg, Sweden |
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| Abstract: | A combination of site-directed mutagenesis and NMR chemical shift perturbation analysis of backbone and side-chain protons has been used to characterize the transient complex of the photosynthetic redox proteins plastocyanin and cytochrome f. To elucidate the importance of charged residues on complex formation, the complex of cytochrome f and E43Q/D44N or E59K/E60Q spinach plastocyanin double mutants was studied by full analysis of the (1)H chemical shifts by use of two-dimensional homonuclear NMR spectra. Both mutants show a significant overall decrease in chemical shift perturbations compared with wild-type plastocyanin, in agreement with a large decrease in binding affinity. Qualitatively, the E43Q/D44N mutant showed a similar interaction surface as wild-type plastocyanin. The interaction surface in the E59K/E60Q mutant was distinctly different from wild type. It is concluded that all four charged residues contribute to the affinity and that residues E59 and E60 have an additional role in fine tuning the orientation of the proteins in the complex. |
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| Keywords: | Plastocyanin cytochrome f chemical shift protein-protein interaction site-directed mutagenesis |
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