Secondary structure of the 33 kDa, extrinsic protein of Photosystem II: a far-UV circular dichroism study.

The 33 kDa extrinsic protein of Photosystem II is an important component of the oxygen-evolving apparatus which functions to stabilize the manganese cluster at physiological chloride concentrations and to lower the calcium requirement for oxygen evolution. Chou-Fasman analysis of the amino-acid sequence of this protein suggests that this component contains a high proportion of alpha-helical structure and only relatively small amounts of beta-sheet structure. A computational study using more sophisticated techniques (Beauregard, M. (1992) Environ. Exp. Bot. 32, 411-429) concluded that the protein contained little periodically ordered secondary structure. In this study, we have directly measured the relative proportions of secondary structure present in the 33 kDa protein using far-ultraviolet circular dichroism spectroscopy. Our results indicate that, in solution, this protein contains a large proportion of beta-sheet structure (38%) and relatively small amounts of alpha-helical structure (9%). A structural model of the 33 kDa protein based on a constrained Chou-Fasman analysis (Teeter, M.M. and Whitlow, M (1988) Proteins 4, 262-273) is presented.

Kinetic characterization of His-tagged CP47 photosystem II in Synechocystis sp. PCC6803.

Recently, construction of strains of Synechocystis sp. PCC6803 having a His(6) extension (His-tag) of the carboxyl terminus of the CP47 protein has been reported (T.M. Bricker et al, Biochim. Biophys. Acta 1409 (1998) 50; M.J. Reifler et al., in: Garab, Pusztai (Eds.) Proc. XIth International Congress on Photosynthesis, 1998). While these initial reports suggest a minimal impact of the His-tag upon Photosystem (PS) II function, a more thorough analysis of the kinetic properties of the modified complex is essential. This communication reports on a more detailed kinetic analysis to assess possible perturbations of PS II due to the genetic addition of the His-tag on the CP47 protein. It was found that: (1) Patterns of flash O(2) yield exhibited normal period four oscillations and the associated fits of the Kok-Joliot S-state cycling parameters were virtually identical to the wild type; (2) O(2) release kinetics during the S(3)-S(0) transition were experimentally indistinguishable from the wild type; (3) S-state decay measurements indicate slightly faster decays of the S(2) and S(3) states compared to the wild type; (4) fluorescence measurements indicate that the kinetics of the forward reaction of electron transfer from Q(A)(-) to Q(B) and back-reactions of Q(A)(-) with PS II electron donors are similar in the His-tag and wild-type strains. It is therefore concluded that the addition of the His-tag results in a minimal perturbation of PS II function.

Isolation of lumenal proteins from spinach thylakoid membranes by triton X-114 phase partitioning.

The proteins present in the thylakoid lumen of higher plant chloroplasts have not been rigorously examined. In this communication we present a simple and rapid procedure for the isolation of the soluble proteins and extrinsic membrane proteins present in the thylakoid lumen from spinach. Our procedure involves extensive washing of the thylakoid membranes followed by Triton X-114 phase partitioning. When analyzed by one-dimensional polyacrylamide gel electrophoresis (PAGE), we obtain results which are very similar to those obtained by Kieselbach et al. using more classical methods [T. Kieselbach, A. Hagman, B. Andersson, W.P. Schroder, J. Biol. Chem. 273 (1998) 6710-6716]. About 25 major proteins are observed upon Coomassie blue staining. Upon two-dimensional isoelectric focusing-sodium dodecyl sulfate-PAGE and either Coomassie blue or silver staining, however, numerous other protein components are resolved. Our findings indicate that the total number of proteins (soluble and extrinsic membrane) present in the lumen may exceed 150.

Isolation of a highly active photosystem II preparation from Synechocystis 6803 using a histidine-tagged mutant of CP 47.

Site-directed mutagenesis was used to produce a Synechocystis mutant containing a histidine tag at the C terminus of the CP 47 protein of Photosystem II. This mutant cell line, designated HT-3, exhibited slightly above normal rates of oxygen evolution and appeared to accumulate somewhat more Photosystem II reaction centers than a control strain. A rapidly isolatable (<7 h) oxygen-evolving Photosystem II preparation was prepared from HT-3 using dodecyl-beta-d-maltoside solubilization and Co2+ metal affinity chromatography. This histidine-tagged Photosystem II preparation stably evolved oxygen at a high rate (2440 micromol O2 (mg chl)-1 h-1), exhibited an alpha-band absorption maximum at 674 nm, and was highly enriched in a number of Photosystem II components including cytochrome c550. Fluorescence yield analysis using water or hydroxylamine as an electron donor to the Photosystem II preparation indicated that virtually all of the Photosystem II reaction centers were capable of evolving oxygen. Proteins associated with Photosystem II were highly enriched in this preparation. 3,3',5, 5'-Tetramethylbenzidine staining indicated that the histidine-tagged preparation was enriched in cytochromes c550 and b559 and depleted of cytochrome f. This result was confirmed by optical difference spectroscopy. This histidine-tagged Photosystem II preparation may be very useful for the isolation of Photosystem II preparations from mutants containing lesions in other Photosystem II proteins.

Epitope mapping of the monoclonal antibody FAC2 on the apoprotein of Cpa-1 in photosystem II.

Using a combination of cyanogen bromide cleavage and endoproteinase digestion we have shown that the putative epitope for the monoclonal antibody FAC2 lies in the region 360Pro(-391)Ser on the apoprotein of CPa-1. This region lies entirely within the large extrinsic loop of this protein. We have shown previously that the epitope of FAC2 becomes exposed in oxygen-evolving membranes upon treatment with alkaline Tris which releases all four of the manganese associated with the oxygen-evolving site of photosystem II. The epitope is not exposed, however, after CaCl(2) treatment and exposure to low concentrations of chloride, conditions which lead to the release of two of the four manganeses associated with the oxygen-evolving site. These results suggest that, upon release of the chloride-insensitive manganese from photosystem II membranes, a conformational change occurs which leads to the exposure of 360Pro(-391)Ser on CPa-1 to the monoclonal antibody FAC2.

The structure and function of CPa-1 and CPa-2 in Photosystem II.

This review presents a summary of recent investigations examining the structure and function of the chlorophyll-proteins CPa-1 (CP47) and CPa-2 (CP43). Comparisons of the derived amino acid sequences of these proteins suggest sites for chlorophyll binding and for interactions between these chlorophyll-proteins and other Photosystem II components. Hydropathy plot analysis of these proteins allows the formulation fo testable hypotheses concerning their topology and orientation within the photosynthetic membrane. The role of these chlorophyll-proteins as interior light-harvesting chlorophyll-a antennae for Photosystem II is examined and other possible additional roles for these important Photosystem II components are discussed.

Oxygen evolution in the absence of the 33-kilodalton manganese-stabilizing protein.

There has been a considerable amount of controversy concerning the ability of photosystem II to evolve oxygen in the absence of the 33-kDa, manganese-stabilizing protein. Early reports indicated that some capacity for oxygen evolution existed in manganese-stabilizing protein-depleted membranes while more recent studies have suggested that the observed oxygen evolution activity arose from residual manganese-stabilizing protein present in the salt-washed preparations. In this paper, it is conclusively demonstrated that significant rates of steady-state oxygen evolution are observed in oxygen-evolving photosystem II membranes in the absence of detectable quantities of the manganese-stabilizing protein. More then 99% of the manganese-stabilizing protein was removed by either one CaCl2 or two NaCl-urea washes. The amount of manganese-stabilizing protein removed was quantified immunologically using mouse polyclonal antibodies. Oxygen evolution rates of 115-140 mumol of O2 (mg of Chl)-1 h-1 were observed in the NaCl-urea-washed preparations. These rates represent about 24% of the rate observed in untreated membranes [450-600 mumol of O2 (mg of Chl)-1 h-1]. Somewhat lower, although still significant rates were observed in the CaCl2-washed preparations. Optimal rates of oxygen-evolving activity in NaCl-urea-washed membranes which are devoid of the manganese-stabilizing protein required high concentrations of calcium and chloride.

Dodecyl maltoside-sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis of chloroplast thylakoid membrane proteins.

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-beta-D-maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-beta-D-maltoside and separated in 4-7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-beta-D-maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5-20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3',5,5'-tetramethylbenzidine-H2O2, Coomassie blue, and silver staining. A number of protein components were identified on "Western blots" of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a/b protein complex, photosystem I, photosystem II, the cytochrome b6/f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.

Site-directed mutagenesis of the CP47 protein of photosystem II: alteration of the basic residue 448R to 448G prevents the assembly of functional photosystem II centers under chloride-limiting conditions.

The psbB gene encodes the intrinsic chlorophyll protein CP47 (CPa-1), a component of photosystem II in higher plants, algae, and cyanobacteria. Oligonucleotide-directed mutagenesis has been used to introduce mutations into a segment of the psbB gene which encodes the large extrinsic loop E of CP47 in the cyanobacterium Synechocystis sp. PCC 6803. One mutation, R448G, produced a strain with impaired photosystem II activity. When grown in standard BG-11 media (480 microM chloride), this strain grew photoautotrophically at about 50% the rate of control strains and exhibited 63% of the control photosystem II activity. Quantum yield measurement at low light intensities indicated that this mutant had 55% of the fully functional photosystem II centers contained in control strains of Synechocystis. Upon exposure to high light intensities, the mutant strain exhibited a 2.2-fold increase in the rate of photoinactivation. When grown in BG-11 which was depleted in chloride (20 microM chloride), the mutant strain exhibited dramatically altered characteristics. Little or no growth was observed in the mutant while the control strains grew at nearly normal rates. Growth rates of the mutant strain could be restored by the addition of 480 microM bromide to the chloride-deficient BG-11 media. In the presence of glucose, the mutant and control strains grew at comparable rates under either chloride-sufficient or chloride-limiting conditions. Analysis of the mutant cell line grown in the absence of chloride and in the presence of glucose indicated that it exhibited essentially no capacity for oxygen evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural organization of proteins on the oxidizing side of photosystem II. Two molecules of the 33-kDa manganese-stabilizing proteins per reaction center.

The 33-kDa manganese-stabilizing protein stabilizes the manganese cluster in the oxygen-evolving complex. There has been, however, a considerable amount of controversy concerning the stoichiometry of this photosystem II (PS II) component. In this paper, we have verified the extinction coefficient of the manganese-stabilizing protein by amino acid analysis, determined the manganese content of oxygen-evolving photosystem II membranes and reaction center complex using inductively coupled plasma spectrometry, and determined immunologically the amount of the manganese-stabilizing protein associated with photosystem II. Oxygen-evolving photosystem II membranes and reaction center complex preparations contained 258 +/- 11 and 67 +/- 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 +/- 0.2 and 2.0 +/- 0.3 molecules of the manganese-stabilizing protein/tetranuclear manganese cluster in oxygen-evolving PS II membranes and highly purified PS II 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 CaCl2-washed PS II membrane proteins or reaction center complex proteins in the manganese-stabilizing 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 II preparations.

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide.

The structural organization of photosystem II 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 II membranes were treated with 1-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 CaCl2 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 manganese-stabilizing 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-terminal 8-kDa peptide of the manganese-stabilizing protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains on CPa-1 which are shielded from N-hydroxysuccinimide biotinylation by the manganese-stabilizing protein.

The structural organization of photosystem II proteins has been investigated by use of the amino group-labeling reagent N-hydroxysuccinimidobiotin (NHS-biotin) and calcium chloride-washed photosystem II membranes. We have previously shown that the presence of the extrinsic, manganese-stabilizing protein on photosystem II membranes prevents the modification of lysyl residues located on the chlorophyll protein CPa-1 (CP-47) by NHS-biotin [Bricker, T. M., Odom, W. R., & Queirolo, C. B. (1988) FEBS Lett. 231, 111-117]. Upon removal of the manganese-stabilizing protein by calcium chloride-washing, CPa-1 can be specifically modified by treatment with NHS-biotin. Preparative quantities of biotinylated CPa-1 were subjected to chemical cleavage with cyanogen bromide. Two major biotinylated peptides were identified with apparent molecular masses of 11.8 and 15.7 kDa. N-terminal sequence analysis of these peptides indicated that the 11.8-kDa peptide was 232G-330M and that the 15.7-kDa peptide was 360P-508V. The 15.7-kDa CNBr peptide was subjected to limited tryptic digestion. The two smallest tryptic fragments identified migrated at apparent molecular masses of 9.1 (nonbiotinylated) and 7.5 kDa (biotinylated). N-terminal sequence analysis and examination of the predicted amino acid sequences of these peptides suggest that the 9.1-kDa fragment was 422R-508V and that the 7.5-kDa fragment was 360P-421A. These results strongly suggest that two NHS-biotinylated domains, 304K-321K and 389K-419K, become exposed on CPa-1 when the manganese-stabilizing protein is removed by CaCl2 treatment. Both of these domains lie in the large extrinsic loop E of CPa-1.

Site-directed mutagenesis of the CPa-1 protein of photosystem II: alteration of the basic residue pair 384,385R to 384,385G leads to a defect associated with the oxygen-evolving complex.

The psbB gene encodes the intrinsic chlorophyll-a binding protein CPa-1 (CP-47), a component of photosystem II in higher plants, algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into a segment of the psbB gene encoding the large extrinsic loop region of CPa-1 in the cyanobacterium Synechocystis sp. PCC 6803. Altered psbB genes were introduced into a mutant recipient strain (DEL-1) of Synechocystis in which the genomic psbB gene had been partially deleted. Initial target sites for mutagenesis were absolutely conserved basic residue pairs occurring within the large extrinsic loop. One mutation, RR384385GG, produced a strain with impaired photosystem II activity. This strain exhibited growth characteristics comparable to controls. However, at saturating light intensities this mutant strain evolved oxygen at only 50% of the rate of the control strains. Quantum yield measurements at low light intensities indicated that the mutant had 30% fewer fully functional photosystem II centers than do control strains of Synechocystis. Immunological analysis of a number of photosystem II protein components indicated that the mutant accumulates normal quantities of photosystem II proteins and that the ratio of photosystem II to photosystem I proteins is comparable to that found in control strains. Upon exposure to high light intensities the mutant cells exhibited a markedly increased susceptibility to photoinactivation. However, Tris-treated thylakoid membranes from both the mutant and wild-type exhibited comparable rates of photoinactivation. Thylakoid membranes isolated from RR384385GG exhibited only 15% of the H2O to 2,6-dichlorophenolindophenol electron transport rate observed in wild-type strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of a monoclonal antibody in structural investigations of the 49-kDa polypeptide of photosystem II.

A monoclonal antibody, FAC2, was isolated by immunization of mice with a Photosystem II core preparation followed by splenic fusion and standard monoclonal antibody screening and production techniques. This antibody recognizes the 49-kDa polypeptide of Photosystem II which is the apoprotein of CPal. The antigenic determinant recognized by this antibody lies on a cyanogen bromide fragment which appears as a doublet with an apparent molecular mass of 14.5 kDa. FAC2 was used to follow the effects of trypsin on the 49-kDa polypeptide in a membrane environment. Our results indicate that the extrinsic polypeptides of Photosystem II which are known to be involved in oxygen evolution protect the 49-kDa polypeptide from tryptic attack. Additionally, Photosystem II membranes which are treated with alkaline Tris exhibit a large increase in the ability to bind FAC2. This increase is not observed with membranes treated with calcium chloride or sodium chloride. These results indicate that the 49-kDa polypeptide may be at least structurally associated with the component(s) responsible for oxygen evolution.

Site-directed mutagenesis of the basic residues 321K to 321G in the CP 47 protein of photosystem II alters the chloride requirement for growth and oxygen-evolving activity in Synechocystis 6803.

CP 47, a component of photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbB gene. Site-specific mutagenesis has been used to alter a portion of the psbB gene encoding the large extrinsic loop E of CP 47 in the cyanobacterium Synechocystis 6803. Alteration of a lysine residue occurring at position 321 to glycine produced a strain with altered PSII activity. This strain grew at wild-type rates in complete BG-11 media (480 microM chloride). However, oxygen evolution rates for this mutant in complete media were only 60% of the observed wild-type rates. Quantum yield measurements at low light intensities indicated that the mutant had 66% of the fully functional PSII centers contained in the control strain. The mutant proved to be extremely sensitive to photoinactivation at high light intensities, exhibiting a 3-fold increase in the rate of photoinactivation. When this mutant was grown in media depleted of chloride (30 microM chloride), it lost the ability to grow photoautotrophically while the control strain exhibited a normal rate of growth. The effect of chloride depletion on the growth rate of the mutant was reversed by the addition of 480 microM bromide to the chloride-depleted BG-11 media. In the presence of glucose, the mutant and control strains grew at comparable rates in either chloride-containing or chloride-depleted media. Oxygen evolution rates for the mutant were further depressed (28% of control rates) under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. Measurements of the variable fluorescence yield indicated that the mutant assembled fewer functional centers in the absence of chloride. These results indicate that the mutation K321G in CP 47 affects PSII stability and/or assembly under conditions where chloride is limiting.

Interaction of the 33 kDa extrinsic protein with photosystem II: rebinding of the 33 kDa extrinsic protein to photosystem II membranes which contain four, two, or zero manganese per photosystem II reaction center.

The 33 kDa extrinsic protein of photosystem II acts to enhance oxygen evolution and to stabilize the manganese cluster at low chloride concentrations. Due to controversies concerning the stoichiometry of this protein [Miyao, M., & Murata, N. (1989) Biochim. Biophys. Acta 977, 315-321, versus Xu, Q., & Bricker, T. M. (1992) J. Biol. Chem. 267. 25816-25821] we have examined the rebinding of this protein to PS II membrane preparations which contain four, two, or zero manganese per photosystem II reaction center. After rebinding, immunoquantification of the 33 kDa extrinsic protein demonstrated that each of these photosystem II membrane preparations strongly bound two copies of the 33 kDa extrinsic protein per photosystem II reaction center. The first and second stoichiometric binding constants (Ka1 and Ka2) for the binding of the 33 kDa protein to PS II centers containing four manganese were 0.42 and 0.67 nM(-1), respectively. Disruption of the manganese cluster either by removal of the chloride-sensitive manganese or extraction of the manganese cluster by alkaline Tris led to a 5-6-fold decrease in Ka1 and about a 3-fold decrease in Ka2. In all cases the binding of the two copies of the 33 kDa extrinsic protein exhibited positive cooperativity with Hill coefficients ranging from 1.6 to 2.2. These findings demonstrate that damage to the manganese cluster alters the binding affinity of the 33 kDa extrinsic protein to photosystem II but does not alter the molecularity of the binding reaction.

Interaction of the 33-kDa extrinsic protein with photosystem II: identification of domains on the 33-kDa protein that are shielded from NHS-biotinylation by photosystem II.

The structural association of the spinach 33-kDa extrinsic protein of photosystem II with the membrane-bound components of the photosystem was investigated by labeling the 33-kDa extrinsic protein with the amino group-specific reagent N-hydroxysuccinimidobiotin both on NaCl-washed photosystem II membranes and free in solution. After quenching of the labeling reagent and isolation of the biotinylated molecules, the biotinylation sites were identified by Staphylococcus V8 protease digestion and analysis of the resultant peptide fragment mixture by matrix-assisted laser desorption/ionization mass spectrometry. When the 33-kDa extrinsic protein was modified on PS II membranes, three domains were biotinylated: 14K, 41K-76K, and 190K-236K. When the 33-kDa extrinsic protein was modified in solution, four additional domains were biotinylated: 1E-4K, 20K, 101K-105K, and 159K-186K. These additional modified domains reside in portions of the 33-kDa protein that are not accessible to the bulk solvent when the protein is associated with PS II and may define regions of interaction with the photosystem.

Triton X-114 phase fractionation of membrane proteins of the cyanobacterium Anacystis nidulans R2.

The thylakoid polypeptides of the cyanobacterium Anacystis nidulans R2 were analyzed by Triton X-114 phase fractionation [C. Bordier (1981) J. Biol. Chem. 256, 1604-1607, as adapted for photosynthetic membranes by T.M. Bricker and L.A. Sherman (1982) FEBS Lett. 149, 197-202]. In this procedure, polypeptides with extensive hydrophobic regions (i.e., intrinsic proteins) form mixed micelles with Triton X-114, and are separated from extrinsic proteins by temperature-mediated precipitation of the mixed Triton X-114-intrinsic protein micelles. The polypeptide pattern after phase fractionation was highly complementary, with 62 of the observed 110 polypeptide components partitioning into the Triton X-114-enriched fraction. Identified polypeptides fractionating into the Triton X-114 phase included the apoproteins for Photosystems I and II, cytochromes f and b6, and the herbicide-binding protein. Identified polypeptides fractioning into the Triton X-114-depleted (aqueous) phase included the large and small subunits of RuBp carboxylase, cytochromes c550 and c554, and ferredoxin. Enzymatic radioiodination of the photosynthetic membranes followed by Triton X-114 phase fractionation allowed direct identification of intrinsic polypeptide components which possess surface-exposed regions susceptible to radioiodination. The most prominent of these polypeptides was a 34-kDa component which was associated with photosystem II. This phase partitioning procedure has been particularly helpful in the clarification of the identity of the membrane-associated cytochromes, and of photosystem II components. When coupled with surface-probing techniques, this procedure is very useful in identifying intrinsic proteins which possess surface-exposed domains. Phase fractionation, in conjunction with the isolation of specific membrane components and complexes, has allowed the identification of many of the important intrinsic thylakoid membrane proteins of A. nidulans R2.

Effects of trypsin and calcium chloride on signal IIs in oxygen-evolving PS II preparations.

Photosystem II oxygen-evolving preparations exhibited a reversible loss of signal IIs hyperfine structure when treated with 1.0 M CaCl2. A progressive irreversible loss of hyperfine structure was observed upon trypsin treatment of these preparations. These treatments appear to alter the environment of the radical responsible for signal IIs. Gel electrophoresis of trypsin-treated photosystem II preparations indicates that three polypeptides (45, 32-34, and 26 kDa) are altered with the same kinetics as observed for the trypsin-induced loss of signal IIs. Two of these polypeptides (45 and 32-34 kDa) are core components of photosystem II.