title: Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding.
abstract: The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c₆ in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1-79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His₆ tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I. and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50-65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and ¹⁵N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.
authors.affiliation: Department of Chemistry, University of Gothenburg, P.O. Box 462, SE-40330 Gothenburg, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Farkas
authors.forename: Lars-Gunnar
authors.initials: LG
authors.lastname: Franzén
authors.forename: Örjan
authors.initials: Ö
authors.lastname: Hansson
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D045365:Cytochromes c6 / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D010446:Peptide Fragments; D045331:Photosystem I Protein Complex / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010970:Plastocyanin / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D018724:Spinacia oleracea / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D013696:Temperature; D013879:Thioredoxins
chemical_list: D045365:Cytochromes c6; D004220:Disulfides; D010446:Peptide Fragments; D045331:Photosystem I Protein Complex; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D010970:Plastocyanin