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. 2006 Dec 26;45(51):15633-43.
doi: 10.1021/bi062148m. Epub 2006 Dec 8.

Micelle-induced folding of spinach thylakoid soluble phosphoprotein of 9 kDa and its functional implications

Jikui Song  1 , Min S Lee, Inger Carlberg, Alexander V Vener, John L Markley
Affiliations

Micelle-induced folding of spinach thylakoid soluble phosphoprotein of 9 kDa and its functional implications

Jikui Song et al. Biochemistry. .

Abstract

Thylakoid soluble phosphoprotein of 9 kDa (TSP9) has been identified as a plant-specific protein in the photosynthetic thylakoid membrane (Carlberg et al. (2003) Proc. Natl. Acad. Sci. 100, 757-762). Nonphosphorylated TSP9 is associated with the membrane, whereas, after light-induced phosphorylation, a fraction of the phosphorylated TSP9 is released into the aqueous stroma. By NMR spectroscopy, we have determined the structural features of nonphosphorylated TSP9 both in aqueous solution and in membrane mimetic micelles. The results show that both wild type nonphosphorylated TSP9 and a triple-mutant (T46E + T53E + T60E) mimic of the triphosphorylated form of TSP9 are disordered under aqueous conditions, but adopt an ordered conformation in the presence of detergent micelles. The micelle-induced structural features, which are similar in micelles either of SDS or dodecylphosphocholine (DPC), consist of an N-terminal alpha-helix, which may represent the primary site of interaction between TSP9 and binding partners, and a less structured helical turn near the C-terminus. These structured elements contain mainly hydrophobic residues. NMR relaxation data for nonphosphorylated TSP9 in SDS micelles indicated that the molecule is highly flexible with the highest order in the N-terminal alpha-helix. Intermolecular NOE signals, as well as spin probe-induced broadening of NMR signals, demonstrated that the SDS micelles contact both the structured and a portion of the unstructured regions of TSP9, in particular, those containing the three phosphorylation sites (T46, T53, and T60). This interaction may explain the selective dissociation of phosphorylated TSP9 from the membrane. Our study presents a structural model for the role played by the structured and unstructured regions of TSP9 in its membrane association and biological function.

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Figures

Figure 1
Figure 1
(A) 1H-15N HSQC spectra of TSP9 in aqueous solution (peaks in black) and in 75 mM SDS (peaks in red). (B) 1H-15N HSQC spectra of (T46E+T53E+T60E) TSP9 in aqueous solution (peaks in black) and in 75 mM SDS (peaks in red). Residues exhibiting significant chemical shift changes on addition of SDS are labeled with their sequence number. The numbering system used here is that of the native, mature sequence of TSP9.
Figure 2
Figure 2
Sequential NOE connectivities used in calculating the structure of TSP9.
Figure 3
Figure 3
Solution structure of TSP9 in SDS micelles. (A) Ribbon representation of the whole TSP9 molecule. The α1, α2, and residues F28–D32 and G59–S62, which show the dαN(i, i+2) NOE connectivities, are green. The structurally disordered regions are shown in light gray. (B) Representation of the two helical structural elements. The figure was made with MOLMOL (75).
Figure 4
Figure 4
(A) Kyte-Doolittle hydropathy plot (76) of TSP9. (B) Selected region of the 15N,13C-filtered/13C-edited 1H,1H-NOESY of TSP9 in SDS micelles, highlighting the NOE signals between the Hγ atoms (QG1) of Val15 (top) and the atoms of SDS molecules (right, C-1H). (C) Residues of TSP9 that show NOEs with SDS micelles are highlighted in dark in the amino acid sequence.
Figure 5
Figure 5
Paramagnetic relaxation of TSP9 induced by nitroxide radicals. (A) Remaining amplitude of the amide peaks of TSP9 after the addition of 5-DSA. (B) Remaining amplitude of the amide peaks of TSP9 after the addition of 16-DSA.
Figure 6
Figure 6
Backbone 15N-R1, R2 relaxation parameters and 1H-15N NOEs of TSP9 in SDS micelles.
Figure 7
Figure 7
Model for the phosphorylation-modulated reversible membrane association of TSP9. The thylakoid membrane is shown by the blue rectangle, and the border in dark blue represents the polar groups of the lipids. The multisubunit PSII complex is represented by the gray (core) and purple blocks, and LHCII is represented by yellow blocks. Phosphorylated threonines are represented by the letter “P” circled in red.
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