Amino acid sequences and solution structures of manganese stabilizing protein that affect reconstitution of Photosystem II activity (original) (raw)
Related papers
Photosynthesis Research, 2005
The Photosystem II (PS II) manganese stabilizing protein (MSP) possesses characteristics, including thermostability, ascribed to the natively unfolded class of proteins (Lydakis-Simantiris et al. (1999) Biochemistry 38: 404-414). A site-directed mutant of MSP, C28A, C51A, which lacks the -SAS-bridge, also binds to PS II at wild-type levels and reconstitutes oxygen evolution activity Biochim Biophys Acta 1274: 135-142], although the mutant protein is even more disordered in solution. Both WT and C28A, C51A MSP aggregate upon heating, but an examination of the effects of protein concentration and pH on heat-induced aggregation showed that each MSP species exhibited greater resistance to aggregation at a pH near their pI (5.2) than do either bovine serum albumin (BSA) or carbonic anhydrase, which were used as model water soluble proteins. Increases in pH above the pI of the MSPs and BSA enhanced their aggregation resistance, a behavior which can be predicted from their charge (MSP) or a combination of charge and stabilization by -SAS-bonds (BSA). In the case of aggregation resistance by MSP, this is likely to be an important factor in its ability to avoid unproductive self-association reactions in favor of formation of the protein-protein interactions that lead to formation of the functional oxygen evolving complex. Abbreviations: BSA -bovine serum albumin; CD -circular dichroism; IPTG -isopropyl-b-D D -thiogalactopyranoside; MSP -manganese stabilizing protein; OEC -O 2 -evolving complex; PAGE -polyacrylamide gel electrophoresis; PS -Photosystem; psbO -gene encoding precursor MSP; WT -wild-type Photosynthesis Research (2005) 84: 283-288 Ó Springer 2005
Biochemistry, 2003
The N-terminus of spinach photosystem II manganese stabilizing protein (MSP) contains two amino acid sequences, 4 KRLTYD 10 E and 15 TYL 18 E, that are necessary for binding of two copies of this subunit to the enzyme [Popelkova et al. (2002) Biochemistry 41, 10038-10045]. To better understand the basis of MSP-photosystem II interactions, the role of threonine residues in the highly conserved motifs T(Y/F)DE and TY has been characterized. Deletion mutants lacking the first 5, 6, 7, and 15 amino acid residues at the N-terminus of the protein were examined for their ability to reconstitute activity in MSP-depleted photosystem II. The results reported here show that truncations of five and six amino acid residues (mutants ∆R5M and ∆L6M mutants) have no negative effect on recovery of oxygen evolution activity or on binding of MSP to photosystem II. Deletion of seven residues (mutant ∆T7M) decreases reconstitution activity to 40% of the control value and reduces functional binding of the mutant protein to photosystem II from two to one copy. Deletion of 15 amino acid residues (mutant ∆T15M) severely impairs functional binding of MSP, and lowers O 2 evolution activity to less than 20% of the control. ∆T7M is the only mutant that exhibited neither nonspecific binding to photosystem II nor changes in tertiary structure. These, and previous results, show that the highly conserved Thr7 and Thr15 residues of MSP are required for functional binding of two copies of the eukaryotic protein to photosystem II. Although the N-terminal domains, 1 EGGKR 6 L, 8 YDEIQS 14 K, and 16 YL 18 E of spinach MSP are unnecessary for specific, functional binding interactions, these sequences are necessary to prevent nonspecific binding of the protein to photosystem II. stabilizing protein; OEC, O2 evolving complex; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; TOPO, plasmid for gene expression with T7 RNA polymerase; PS, photosystem; psbO, gene encoding precursor MSP; SDS, sodium dodecyl sulfate; sw-PSII, NaCl-washed photosystem II membranes depleted of 23 and 17 kDa extrinsic proteins; TMACl, tetramethylamonium chloride; usw-PSII, urea salt-washed photosystem II membranes depleted of 33, 23, and 17 kDa extrinsic proteins; UV, ultraviolet; ∆, represents missing amino acid residues; 2°, secondary structure of protein; 3°, tertiary structure of protein.
Manganese Stabilizing Protein of Photosystem II Is a Thermostable, Natively Unfolded Polypeptide †
Biochemistry, 1999
The thermostability of manganese stabilizing protein of photosystem II was examined by biochemical and spectroscopic techniques. Samples of both native and recombinant spinach manganese stabilizing protein incubated at 90°C and then cooled to 25°C were capable of rebinding to, and of reactivating, the O 2 -evolution activity of photosystem II membranes from which the native protein had been removed. Far-UV circular dichroism and FT-IR spectroscopies were used to analyze the structural consequences of heating manganese stabilizing protein. The data obtained from these techniques show that heating causes a complete loss of the protein's secondary structure, and that this is a reversible, noncooperative phenomenon. Upon cooling, the secondary structures of the heat-treated proteins return to a state similar to, but not identical with, that of the native, unheated controls. Restoration of a nearnative tertiary structure is confirmed both by size-exclusion chromatography and by near-UV circular dichroism. The functional and structural thermostability of manganese stabilizing protein reported here, in conjunction with additional known properties of this protein (acidic pI, high random coil and turn content, anomalous hydrodynamic behavior), identifies manganese stabilizing protein as a natively unfolded protein [Weinreb et al. (1996) Biochemistry 35, 13709-13715]. Although these proteins lack amino acid sequence identity, their functional solution conformations under physiological conditions are said to be "natively unfolded". We suggest that, as with other members of this family of proteins, the natively unfolded structure of manganese stabilizing protein facilitates the highly effective protein-protein interactions that are necessary for its assembly into photosystem II.
Biochemistry, 2002
The importance of the N-terminal domain of manganese stabilizing protein in binding to photosystem II has been previously demonstrated [Eaton-Rye and Murata (1989) Biochim. Biophys. Acta 977, 219-226; Odom and Bricker (1992) Biochemistry 31, 5616-5620]. In this paper, we report results from a systematic study of functional and structural consequences of N-terminal elongation and truncation of manganese stabilizing protein. Precursor manganese stabilizing protein is the unprocessed wild-type protein, which carries an N-terminal extension of 84 amino acids in the form of its chloroplastic signal peptide. Despite its increased size, this protein is able to reconstitute O 2 evolution activity to levels observed with the mature, processed protein, but it also binds nonspecifically to PSII. Truncation of wild-type manganese stabilizing protein by site-directed mutagenesis to remove three N-terminal amino acids, resulting in a mutant called ∆G3M, causes no loss of activity reconstitution, but this protein also exhibits nonspecific binding. Further truncation of the wild-type protein by ten N-terminal amino acids, producing ∆E10M, limits binding of manganese stabilizing protein to 1 mol/mol of photosystem II and decreases activity reconstitution to about 65% of that obtained with the wild-type protein. Because two copies of wild type normally bind to photosystem II, amino acids in the domain 4 K-10 E must be involved in the binding of one copy of manganese stabilizing protein to photosystem II. Spectroscopic analysis (CD and UV spectra) reveals that N-terminal elongation and deletion of manganese stabilizing protein influence its overall conformation, even though secondary structure content is not perturbed. Our data suggest that the solution structure of manganese stabilizing protein attains a more compact solution structure upon removal of N-terminal amino acids.
Biochemistry, 1992
The structural organization of photosystem I1 proteins has been investigated by use of the zero-length protein cross-linking reagent 1 -ethyl-3-[ 3-(dimethylamino)propyl]carbodiimide and monoclonal and polyclonal antibody reagents. Photosystem I1 membranes were treated with l-ethyl-3-[3-(dimethylamino) propyl] carbodiimide which cross-links amino groups to carboxyl groups which are in van der Waals contact. This treatment did not affect the oxygen evolution rates of these membranes and increased the retention of oxygen evolution after CaCl, washing. Analysis of the proteins cross-linked by this treatment indicated that two cross-linked species with apparent molecular masses of 95 and 110 kDa were formed which cross-reacted with antibodies against both the 33-kDa manganese-stabilizing protein and the chlorophyll protein CPa-1. Cleavage of the 110-kDa cross-linked species with cyanogen bromide followed by N-terminal sequence analysis was used to identify the peptide fragments of CPa-1 and the manganese-stabilizing protein which were cross-linked. Two cyanogen bromide fragments were identified with apparent molecular masses of 50 and 25 kDa. N-Terminal sequence analysis of the 50-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa fragment of CPa-1 and the intact manganese-stabilizing protein. This strongly suggests that the manganesestabilizing protein is cross-linked to the large extrinsic loop domain of CPa-1. N-Terminal analysis of the 25-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa peptide of CPa-1 and the N-terminal8-kDa peptide of the manganese-stabilizing protein. These results indicate that the domain 3a4E-440D of CPa-1 is cross-linked to the domain 1E-76K of the manganese-stabilizing protein. The large extrinsic loop domain of CPa-1 thus appears to anchor the manganese-stabilizing protein to the photosynthetic membrane via a charge-pair interaction. 161-165. 1060,224-232. 231-234. FEBS Lett. 167, 127-130. Lett. 234, 374-378. 3275-3282. 741-747. Biochim. Biophys. Acta 806, 283-289. Methods 92, 65-71. Lett. 202, 235-239. Biochemistry 28, 5560-5567. Acta 890, 151-159. 3 6 8-3 7 9. 453-457. Acad. Sci. U.S.A. 76, 4350-4354. Plant Mol. Biol. 8, 317-326. 671-678. (1981) FEBS Lett. 133, 265-268.
2004
Key words: manganese stabilizing protein, natively unfolded protein, Photosystem II, thermostability The Photosystem II (PS II) manganese stabilizing protein (MSP) possesses characteristics, including thermostability, ascribed to the natively unfolded class of proteins (Lydakis-Simantiris et al. (1999) Bio-chemistry 38: 404–414). A site-directed mutant of MSP, C28A, C51A, which lacks the-SAS- bridge, also binds to PS II at wild-type levels and reconstitutes oxygen evolution activity [Betts et al. (1996) Biochim Biophys Acta 1274: 135–142], although the mutant protein is even more disordered in solution. Both WT and C28A, C51A MSP aggregate upon heating, but an examination of the effects of protein concentration and pH on heat-induced aggregation showed that each MSP species exhibited greater resistance to aggregation at a pH near their pI (5.2) than do either bovine serum albumin (BSA) or carbonic anhydrase, which were used as model water soluble proteins. Increases in pH above the ...
Biochemistry, 1998
The extrinsic manganese stabilizing protein of photosystem II is required for Mn retention by the O 2 -evolving complex, accelerates the rate of O 2 evolution, and protects photosytem II against photoinhibition. We report results from studies of the in vitro reconstitution of spinach photosytem II with recombinant manganese stabilizing protein with C-terminal deletions of two, three, and four amino acids. The deletions were the result of amber mutations introduced by site-directed mutagenesis. Removal of the C-terminal dipeptide (Glu-Gln) did not diminish the ability of the manganese stabilizing protein either to rebind to or to restore high rates of O 2 evolution to photosystem II preparations depleted of the native protein. Deletion of the C-terminal tripeptide (Leu-Glu-Gln) resulted in weakened but specific binding of manganese stabilizing protein to photosystem II and minimal recovery of O 2 evolution activity.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2001
Exposure of photosystem II membranes to trypsin that has been treated to inhibit chymotrypsin activity produces limited hydrolysis of manganese stabilizing protein. Exposure to chymotrypsin under the same conditions yields substantial digestion of the protein. Further probing of the unusual insensitivity of manganese stabilizing protein to trypsin hydrolysis reveals that increasing the temperature from 4 to 25³C will cause some acceleration in the rate of proteolysis. However, addition of low (100 WM) concentrations of NH 2 OH, that are sufficient to reduce, but not destroy, the photosystem II Mn cluster, causes a change in PS II-bound manganese stabilizing protein that causes it to be rapidly digested by trypsin. Immunoblot analyses with polyclonal antibodies directed against the N-terminus of the protein, or against the entire sequence show that trypsin cleavage produces two distinct peptide fragments estimated to be in the 17^20 kDa range, consistent with proposals that there are 2 mol of the protein/mol photosystem II. The correlation of trypsin sensitivity with Mn redox state(s) in photosystem II suggest that manganese stabilizing protein may interact either directly with Mn, or alternatively, that the polypeptide is bound to another protein of the photosystem II reaction center that is intimately involved in binding and redox activity of Mn. ß
Photosynthesis Research, 2005
The 33-kDa manganese-stabilizing protein (MSP) of Photosystem II (PS II) maintains the functional stability of the Mn cluster in the enzyme's active site. This protein has been shown to possess characteristics similar to those of the intrinsically disordered, or natively unfolded proteins [Lydakis-Simantiris et al. (1999b) Biochemistry 38: 404-414]. Alternately it was proposed that MSP should be classified as a molten globule, based in part on the hypothesis that its lone disulfide bridge is necessary for structural stability and function in solution [Shutova et al. (2000) FEBS Lett. 467: 137-140]. A site-directed mutant MSP (C28A,C51A) that eliminates the disulfide bond reconstitutes O 2 evolution activity and binds to MSP-free PS II preparations at wild-type levels Biochim. Biophys. Acta 1274: 135-142]. This mutant was further characterized by incubation at 90°C to determine the effect of loss of the disulfide bridge on MSP thermostability and solution structure. After heating at 90°C for 20 min, C28A,C51A MSP was still able to bind to PS II preparations at molar stoichiometries similar to those of WT MSP and reconstitute O 2 evolution activity. A fraction of the protein aggregates upon heating, but after resolubilization, it regains the ability to bind to PS II and reconstitute O 2 evolution activity. Characterization of the solution structure of C28A,C51A MSP, using CD spectroscopy, UV absorption spectroscopy, and gel filtration chromatography, revealed that the mutant has a more disordered solution structure than WT MSP. The disulfide bond is therefore unnecessary for MSP function and the intrinsically disordered characteristics of MSP are not dependent on its presence. However, the disulfide bond does play a role in the solution structure of MSP in vivo, as evidenced by the lability of a C20S MSP mutation in Synechocystis 6803 [Burnap et al. (1994) Biochemistry 33: 13712-13718]. composed of sucrose (0.4 M), MES (50 mM, pH 6.0), NaCl (10 mM)); SW-PS II -Photosystem II preparation treated with 2 M NaCl to extract Ca 2+ and the 23 and 17 kDa extrinsic polypeptides; USW-PS II -Photosystem II preparation treated with 2 M NaCl to extract Ca 2+ and the 23 and 17 kDa extrinsic polypeptides, followed by incubation with 3.1 M urea and 240 mM NaCl to remove the manganese-stabilizing protein Photosynthesis Research (2005) 85: 359-372 Ó Springer 2005
Journal of Biological Chemistry
The 33-kDa manganese-stabilizingprotein stabilizes the manganese cluster in the oxygen-evolving complex. There has been, however, a considerable amount of controversy concerning the stoichiometry of this photosystem I1 (PS 11) component. In this paper, we have verified the extinction coefficient of the manganesestabilizing protein by amino acid analysis, determined the manganese content of oxygen-evolving photosystem I1 membranes and reaction center complex using inductively coupled plasma spectrometry, and determined immunologically the amount of the manganesestabilizing protein associated with photosystem 11. Oxygen-evolving photosystem I1 membranes and reaction center complex preparations contained 258 f 11 and 67 f 3 chlorophyll, respectively, per tetranuclear manganese cluster. Immunoquantification of the manganese-stabilizing protein using mouse polyclonal antibodies on "Western blots" demonstrated the presence of 2.1 f 0.2 and 2.0 f 0.3 molecules of the manganese-stabilizing ProteinJtetranuclear manganese cluster in oxygen-evolving PS I1 membranes and highly purified PS I1 reaction center complex, respectively. Since the manganese-stabilizing protein co-migrated with the D2 protein in our electrophoretic system, accurate immunoquantification required the inclusion of CaClz-washed PS I1 membrane proteins or reaction center complex proteins in the manganesestabilizing protein standards to compensate for the possible masking effect of the D2 protein on the binding of the manganese-stabilizing protein to Immobilon-P membranes. Failure to include these additional protein components in the manganese-stabilizing protein standards leads to a marked underestimation of the amount of the manganese-stabilizing protein associated with these photosystem I1 preparations.