Beta-helix core packing within the triple-stranded oligomerization domain of the P22 tailspike.

A right-handed parallel beta-helix of 400 residues in 13 tightly packed coils is a major motif of the chains forming the trimeric P22 tailspike adhesin. The beta-helix domains of three identical subunits are side-by-side in the trimer and make predominantly hydrophilic inter-subunit contacts (Steinbacher S et al., 1994, Science 265:383-386). After the 13th coil the three individual beta-helices terminate and the chains wrap around each other to form three interdigitated beta-sheets organized into the walls of a triangular prism. The beta-strands then separate and form antiparallel beta-sheets, but still defining a triangular prism in which each side is a beta-sheet from a different subunit (Seckler R, 1998, J Struct Biol 122:216-222). The subunit interfaces are buried in the triangular core of the prism, which is densely packed with hydrophobic side chains from the three beta-sheets. Examination of this structure reveals that its packed core maintains the same pattern of interior packing found in the left-handed beta-helix, a single-chain structure. This packing is maintained in both the interdigitated parallel region of the prism and the following antiparallel sheet section. This oligomerization motif for the tailspike beta-helices presumably contributes to the very high thermal and detergent stability that is a property of the native tailspike adhesin.

Leucine 245 is a critical residue for folding and function of the manganese stabilizing protein of photosystem II.

In solution, Manganese Stabilizing Protein, the polypeptide which is responsible for the structural and functional integrity of the manganese cluster in photosystem II, is a natively unfolded protein with a prolate ellipsoid shape [Lydakis-Simantiris et al. (1999) Biochemistry 38, 404-414; Zubrzycki et al. (1998) Biochemistry 37, 13553-13558]. The C-terminal tripeptide of Manganese Stabilizing Protein was shown to be critical for binding to photosystem II and restoration of O(2) evolution activity [Betts et al. (1998) Biochemistry 37, 14230-14236]. Here, we report new biochemical, hydrodynamic, and spectroscopic data on mutants E246K, E246STOP, L245E, L245STOP, and Q244STOP. Truncation of the final dipeptide (E246STOP) or substitution of Glu246 with Lys resulted in no significant changes in secondary and tertiary structures of Manganese Stabilizing Protein as monitored by CD spectroscopy. The apparent molecular mass of the protein remained unchanged, both mutants were able to rebind to photosystem II, and both proteins reactivate O(2) evolution. Manganese Stabilizing Protein lacking the final tripeptide (L245STOP), or substitution of Glu for Leu245 dramatically modified the protein's solution structure. The apparent molecular masses of these mutants increased significantly, which might indicate unfolding of the protein in solution. This was verified by CD spectroscopy. Both mutant proteins rebound to photosystem II with lower affinities, and activation of O(2) evolution was decreased dramatically. Enhancement of these defects was observed upon removal of the final tetrapeptide (Q244STOP). These results indicate that Leu245 is essential to maintaining Manganese Stabilizing Protein's solution structure in a conformation that promotes efficient binding to photosystem II and/or for the subsequent steps that lead to enzyme activation. Based on an analysis of the properties of C-terminal mutations, a hypothesis for structural requirements for functional binding of Manganese Stabilizing Protein to photosystem II is presented. Effects of C-terminal mutations on the UV spectrum of Manganese Stabilizing Protein were also examined. Mutations that alter solution structure also affect a 293 nm absorption shoulder which is assigned to the only tryptophan residue, Trp241, in the protein, and this absorbance feature is shown to be a useful indicator of alterations to the Trp241 environment.

Manganese stabilizing protein of photosystem II is a thermostable, natively unfolded polypeptide.

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 degreesC and then cooled to 25 degreesC were capable of rebinding to, and of reactivating, the O2-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 near-native 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.

The carboxyl-terminal tripeptide of the manganese-stabilizing protein is required for quantitative assembly into photosystem II and for high rates of oxygen evolution activity.

The extrinsic manganese stabilizing protein of photosystem II is required for Mn retention by the O2-evolving complex, accelerates the rate of O2 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 O2 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 O2 evolution activity. Removal of the C-terminal tetrapeptide (Gln-Leu-Glu-Gln) eliminated the ability of the subunit to interact stably with all of its available binding sites on photosystem II, as evidenced by the fact that this mutant was totally inactive in restoring O2 evolution activity. Evidence is presented to indicate that these mutational effects on the binding and function of the manganese stabilizing protein may be due to major changes in tertiary structure. The truncation mutations lacking either the C-terminal tri- or tetrapeptide exhibit apparent size increases of 25 and 40%, respectively, when compared either to a mutant lacking the C-terminal dipeptide or to the wild-type protein.

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly.

Conformational changes in the extrinsic manganese stabilizing protein can occur upon binding to the photosystem II reaction center: an isotope editing and FT-IR study.

Photosystem II catalyzes the light-driven oxidation of water and reduction of plastoquinone in oxygenic photosynthesis. The manganese stabilizing protein (MSP) of photosystem II is an extrinsic subunit that plays an important role in catalytic activity. This subunit can be extracted and re-bound to the photosystem II reaction center. Extraction is associated with decreased stability of manganese binding by the enzyme and by loss in high rates of oxygen evolution activity; reconstitution reverses these phenomena. Since little is known about the assembly of complex membrane proteins, we have employed isotope editing and vibrational spectroscopy to obtain information about any changes in secondary structure that occur in MSP upon functional reconstitution to photosystem II. The spectroscopic data obtained are consistent with substantial changes in conformation when MSP binds to photosystem II; approximately 30-40% of the peptide backbone undergoes a change in secondary structure. These conclusions were reached by comparing different aliquots, before and after binding, of the same 13[C]MSP sample. Analysis of amide I band line shapes through Fourier deconvolution and nonlinear regression suggests that binding of MSP to photosystem II is associated with a decrease in random structure and an increase in beta-sheet content. We conclude that binding of MSP to the reaction center can induce folding of MSP. Our results also indicate that, in solution, MSP can sample a variety of conformational states, which differ in hydrogen bonding of the peptide backbone.

Mutation Val235Ala weakens binding of the 33-kDa manganese stabilizing protein of photosystem II to one of two sites.

The 33-kDa protein of the photosynthetic O2-evolving complex, also known as manganese stabilizing protein, contributes to the structural stability of the photosystem II tetranuclear Mn cluster and stimulates the water-oxidizing activity of this cluster. Quantification of extrinsic polypeptides in photosystem II has yielded data that support stoichiometries of either one or two copies of each protein per photosystem II reaction center. We recently described the cold-sensitive assembly of a mutant 33-kDa protein with a single amino acid replacement (Val235Ala) [Betts, S. D., Ross, J. R., Pichersky, E., & Yocum, C. F. (1996) Biochemistry 35, 6302-6307]. We have extended the characterization of this mutation. When photosystem II membranes depleted of the 33 kDa extrinsic protein are exposed to mixtures of wild type and Val235Ala manganese stabilizing protein, binding of the wild type protein is strongly preferred. If, however, protein containing the Val235Ala mutation is first bound to photosystem II only half of this protein (about 1 mol/mol of photosystem II reaction centers) is susceptible to displacement by the wild type protein, even after multiple exposures to the latter. These results support the conclusion that 2 mol of manganese stabilizing protein are bound per reaction center. Our data show as well that the mutant 33-kDa protein competes with the wild type protein for at least one of two binding sites on photosystem II and that the mutant protein binds tightly to only one of two sites. These results demonstrate that the two binding sites on photosystem II for the 33-kDa protein have different properties with respect to recognition and binding of this protein.

Functional reconstitution of photosystem II with recombinant manganese-stabilizing proteins containing mutations that remove the disulfide bridge.

The 33-kDa extrinsic subunit of PSII stabilizes the O2-evolving tetranuclear Mn cluster and accelerates O2 evolution. We have used site-directed mutagenesis to replace one or both Cys residues in spinach MSP with Ala. Previous experiments using native and reduced MSP led to the conclusion that a disulfide bridge between these two cysteines is essential both for its binding and its functional properties. We report here that the disulfide bridge, though essential for MSP stability, is otherwise dispensible. The mutation C51A by itself had a delayed effect on MSP function: [C51A]MSP restored normal rates of O2 evolution to PSII but was defective in stabilizing this activity during extended illumination. In contrast, the Cys-free double mutant, [C28A,C51A]MSP, was functionally identical to the wild-type protein. Based on results presented here, we propose a light-dependent interaction between MSP and PSII that occurs only during the redox cycling of the Mn cluster and which is destabilized by the single mutation, C51A.

Cold-sensitive assembly of a mutant manganese-stabilizing protein caused by a Val to Ala replacement.

Photosystem II (PSII) is a multisubunit transmembrane protein complex that oxidizes water and evolves O2. A tetranuclear manganese cluster associated with integral membrane subunits of PSII catalyzes water oxidation. The 33-kDa water-soluble PSII subunit, or manganese-stabilizing protein (MSP), stabilizes the O2-evolving manganese cluster and accelerates O2 evolution. Spinach PSII can be depleted of native MSP under conditions which retain a functional manganese cluster. Reconstition of MSP-depleted PSII with recombinant MSP was equally efficient at 4 and 22 degrees C. Replacement of Val235 (a conserved residue near the C-terminus of MSP) with Ala inhibited assembly of MSP at 4 degrees C, but not at 22 degrees C. Once assembled, [V235A]MSP remained bound to PSII even at 4 degrees C and in the presence of low concentrations of urea. Results from far-UV circular dichroism spectrometry indicated that [V235A]-MSP was destabilized by low temperature to a greater extent than the wild-type protein. However, the effect of temperature on the secondary structure of both the mutant and wild-type proteins was small compared to the temperature-independent destabilization of secondary structure induced by the mutation. These results demonstrate that the V235A mutation introduces an activation energy barrier for assembly of MSP into PSII, and it is suggested that the mutation acts by inhibiting isomerization of one or more prolyl peptide bonds required for assembly.

Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein.

The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O2 evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217-227).

Characterization by electron microscopy of isolated particles and two-dimensional crystals of the CP47-D1-D2-cytochrome b-559 complex of photosystem II.

A photosystem II complex containing the reaction center proteins D1 and D2, a 47-kDa chlorophyll-binding protein (CP47), and cytochrome b-559 was isolated with high yield, purity, and homogeneity; small but well-ordered two-dimensional crystals were prepared from the particles. The crystals and the isolated particles were analyzed by electron microscopy using negatively stained specimens. The information of 20 different digitized crystals was combined by alignment programs based on correlation methods to obtain a final average. The calculated diffraction pattern, with spots up to a resolution of 2.5 nm, and the optical diffraction pattern of a single crystal indicate that the plane group is p22121 (also called p2gg) and that the unit cell is rectangular with parameters of 23.5 x 16.0 nm, containing four stain-excluding monomers (two face-up and two face-down). In projection, the monomers have an asymmetrical shape with a length of 10 nm, a maximal width of 7.5 nm, and a height of 6 nm; their molecular mass is 175 +/- 40 kDa.