Surface interactions in the complex between cytochrome f and the E43Q/D44N and E59K/E60Q plastocyanin double mutants as determined by (1)H-NMR chemical shift analysis

Bergkvist, AndersBiochemistry and Biophysics, Department of Chemistry, Göteborg University, Sweden / Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, USA (författare)

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.

Ejdebäck, Mikael,1969-Högskolan i Skövde,Institutionen för naturvetenskap,Biochemistry and Biophysics, Department of Chemistry, Göteborg University, Sweden(Swepub:his)ejdm (författare)
Ubbink, MarcellusLeiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, The Netherlands (författare)
Karlsson, B. GöranDepartment of Molecular Biotechnology, Chalmers University of Technology, Göteborg, Sweden (författare)
Biochemistry and Biophysics, Department of Chemistry, Göteborg University, Sweden / Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, USAInstitutionen för naturvetenskap (creator_code:org_t)

Av författaren/redakt...: Bergkvist, Ander ...; Ejdebäck, Mikael ...; Ubbink, Marcellu ...; Karlsson, B. Gör ...

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