Conformation of a purified "spontaneously" inserting thylakoid membrane protein precursor in aqueous solvent and detergent micelles.
Subunit W of photosystem II (PsbW) is a single-span thylakoid membrane protein that is synthesized with a cleavable hydrophobic signal peptide and integrated into the thylakoid membrane by an apparently spontaneous mechanism. In this study, we have analyzed the secondary structure of the pre-protein at early stages of the insertion pathway, using purified recombinant pre-PsbW. We show that the protein remains soluble in Tris buffer after removal of detergent. Under these conditions pre-PsbW contains no detectable alpha-helix, whereas substantial alpha-helical structure is present in SDS micelles. In aqueous buffer, the tryptophan fluorescence emission characteristics are intermediate between those of solvent-exposed and hydrophobic environments, suggesting the formation of a partially folded structure. If denaturants are excluded from the purification protocol, pre-PsbW purifies instead as a 180-kDa oligomer with substantial alpha-helical structure. Mature-size PsbW was prepared by removal of the presequence, and we show that this protein also contains alpha-helix in detergent but in lower quantities than the pre-protein. We therefore propose that pre-PsbW contains alpha-helical structure in both the mature protein and the signal peptide in nonpolar environments. We propose that pre-PsbW acquires its alpha-helical structure only during the later, membrane-bound stages of the insertion pathway, after which it forms a "helical hairpin"-type loop intermediate in the thylakoid membrane.
Department of Chemistry and Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
C A
CA
Woolhead
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Mant
S J
SJ
Kim
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C
Robinson
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Rodger
D002850:Chromatography, Gel; D002942:Circular Dichroism; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000737:chemistry*; D008823:Micelles; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D045332:Photosystem II Protein Complex; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry*; D012997:Solvents; D014867:Water
D003902:Detergents; D008823:Micelles; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011498:Protein Precursors; D012997:Solvents; D014867:Water