Micelle-Induced Folding of Spinach Thylakoid Soluble Phosphoprotein of 9 kDa and Its Functional Implications^,

Abstract

Thylakoid soluble phosphoprotein of 9 kDa (TSP9) has been identified as a plant-specific protein in the photosynthetic thylakoid membrane (Carlberg et al., Proc. Natl. Acad. Sci. 2003, 100, 757-762). Non-phosphorylated 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 non-phosphorylated TSP9 in both 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 tri-phosphorylated 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 α-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 non-phosphorylated TSP9 in DPC micelles indicated that the molecule is highly flexible with the highest order in the N-terminal α-helix. Intermolecular NOE signals, as well as spin probeinduced 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 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. † This work was supported by NIH grants P50 GM64598 and U54 GM074901 from the National Institute of General Medical Sciences (J.L.M., P.I). NMR data were collected at the National Magnetic Resonance Facility at Madison, which is supported in part by NIH grants P41 RR02301 and P41 GM66326. ‡ Structure determined under the National Institutes of Health, NIGMS Protein Structure Initiative; coordinates and related data have been deposited at PDB (2FFT) and NMR data at BMRB (bmr 6926).* To whom correspondence should be addressed: Department of Biochemistry, University of Madison, WI 53706. Telephone: (608) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe central player in plant photosynthesis is photosystem II (PSII) 1 in the thylakoid membrane (1,2), which absorbs solar energy and catalyzes the splitting of water into dioxgen and reducing equivalents. The light-harvesting complex II (LHCII), peripheral to the PSII, helps to increase the effective energy absorption by PSII (3). The photosynthetic process in this PSII-LHCII multisubunit complex is regulated by a unique light-and redox-controlled protein phosphorylation system (4,5). In response to light, the plastoquinol pool becom...

The Desmoglein-Specific Cytoplasmic Region Is Intrinsically Disordered in Solution and Interacts with Multiple Desmosomal Protein Partners

“…Micelles formed by SDS have been used to mimic membranes and have consistently been shown to induce secondary structure, whereas TFE is widely known to induce structure formation. 34,35 Taken together, the results indicate that the isolated DSCR, while intrinsically disordered in solution, is able to adopt regular structure at physiological temperatures and in the presence of alcohols and micelles and hint that a biological context could also stabilize its nascent structure.…”

The Mobile Thylakoid Phosphoprotein TSP9 Interacts with the Light-harvesting Complex II and the Peripheries of Both Photosystems

“…Several lines of data show that TSP9 is a highly disordered protein, lacking a unique structure under physiological conditions (19). Proteins with high intrinsic disorder form a newly recognized class of proteins (20,21).…”

“…Upon phosphorylation, TSP9 is partially released from the membrane, and we have suggested that it could play a role in chloroplast signaling (18). NMR spectroscopic studies have shown that TSP9 is highly disordered in aqueous solution but adopts a more ordered conformation under membrane mimetic conditions (19). Intrinsically disordered proteins form a growing family of proteins commonly involved in recognition, regulation, and cell signaling that has only recently been recognized, presumably due to a strong bias in classical biochemical methods toward folded proteins (for reviews, see Refs.…”

D2P2: database of disordered protein predictions

“…Regions of IDP can exist as unfolded chains or molten globules with well-developed secondary structure and often function through transition between differently folded states (2). Mechanisms for functional conformational transition include binding with other proteins, nucleic acids, various small molecules and numerous posttranslational modifications, such as phosphorylation, which has been shown to be especially important (3,4). Biological functions of known IDPs are varied and their roles include the following: instigation of protein complex formation, molecular recognition as seen in nucleoporins of the nuclear pore complex (5), signal transduction, transcriptional regulation and many other functions (6,7) too numerous to list here.…”