Conformation of a Purified "Spontaneously" Inserting Thylakoid Membrane Protein Precursor in Aqueous Solvent and Detergent Micelles

Cheryl A. Woolhead, Alexandra Mant, Soo Jung Kim, Colin Robinson, Alison Rodger

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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 α-helix, whereas substantial α-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 α-helical structure. Mature-size PsbW was prepared by removal of the presequence, and we show that this protein also contains α-helix in detergent but in lower quantities than the pre-protein. We therefore propose that pre-PsbW contains α-helical structure in both the mature protein and the signal peptide in nonpolar environments. We propose that pre-PsbW acquires its α-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.

Original languageEnglish (US)
Pages (from-to)14607-14613
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number18
DOIs
StatePublished - May 4 2001
Externally publishedYes

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Thylakoid Membrane Proteins
Protein Precursors
Micelles
Detergents
Conformations
Thylakoids
Protein Sorting Signals
Proteins
Membranes
Secondary Protein Structure
Tromethamine
Photosystem II Protein Complex
Tryptophan
Buffers
Oligomers
Fluorescence
Purification

ASJC Scopus subject areas

  • Biochemistry

Cite this

Conformation of a Purified "Spontaneously" Inserting Thylakoid Membrane Protein Precursor in Aqueous Solvent and Detergent Micelles. / Woolhead, Cheryl A.; Mant, Alexandra; Kim, Soo Jung; Robinson, Colin; Rodger, Alison.

In: Journal of Biological Chemistry, Vol. 276, No. 18, 04.05.2001, p. 14607-14613.

Research output: Contribution to journalArticle

Woolhead, Cheryl A. ; Mant, Alexandra ; Kim, Soo Jung ; Robinson, Colin ; Rodger, Alison. / Conformation of a Purified "Spontaneously" Inserting Thylakoid Membrane Protein Precursor in Aqueous Solvent and Detergent Micelles. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 18. pp. 14607-14613.
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abstract = "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 α-helix, whereas substantial α-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 α-helical structure. Mature-size PsbW was prepared by removal of the presequence, and we show that this protein also contains α-helix in detergent but in lower quantities than the pre-protein. We therefore propose that pre-PsbW contains α-helical structure in both the mature protein and the signal peptide in nonpolar environments. We propose that pre-PsbW acquires its α-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.",
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