Characterization of amyloidogenic intermediate states through a combined use of CD and NMR spectroscopy.

Characterization of amyloidogenic intermediate states is of central importance in understanding the molecular mechanism of amyloid formation. In this study, we utilized CD and NMR spectroscopy to investigate secondary structure of the monomeric amyloidogenic intermediate of a beta-structured SH3 domain, which was induced by trifluoroethanol (TFE). The combined biophysical studies showed that the native state SH3 domain is gradually converted to the amyloidogenic intermediate state at TFE concentrations of 20-26% (v/v) and the aggregation-prone state contains substantial amount of the beta-sheet conformation ( approximately 30%) with disordered (54%) and some helical characters (16%). Under weaker amyloidogenic conditions of higher TFE concentrations (>40%), the beta-sheet structures were gradually changed to helical conformations and the relative content of the helical and beta-sheet conformations was highly correlated with the aggregation propensity of the SH3 domain. This indicates that the beta-sheet characters of the amyloidogenic states may be critical to the effective amyloid formation.

NMR characterizations of an amyloidogenic conformational ensemble of the PI3K SH3 domain.

Amyloid formation is associated with structural changes of native polypeptides to monomeric intermediate states and their self-assembly into insoluble aggregates. Characterizations of the amyloidogenic intermediate state are, therefore, of great importance in understanding the early stage of amyloidogenesis. Here, we present NMR investigations of the structural and dynamic properties of the acid-unfolded amyloidogenic intermediate state of the phosphatidylinositol 3-kinase (PI3K) SH3 domain--a model peptide. The monomeric amyloidogenic state of the SH3 domain studied at pH 2.0 (35 degrees C) was shown to be substantially disordered with no secondary structural preferences. (15)N NMR relaxation experiments indicated that the unfolded polypeptide is highly flexible on a subnanosecond timescale when observed under the amyloidogenic condition (pH 2.0, 35 degrees C). However, more restricted motions were detected in residues located primarily in the beta-strands as well as in a loop in the native fold. In addition, nonnative long-range interactions were observed between the residues with the reduced flexibility by paramagnetic relaxation enhancement (PRE) experiments. These indicate that the acid-unfolded state of the SH3 domain adopts a partly folded conformation through nonnative long-range contacts between the dynamically restricted residues at the amyloid-forming condition.

Mutagenesis of CP43-arginine-357 to serine reveals new evidence for (bi)carbonate functioning in the water oxidizing complex of Photosystem II.

The chlorophyll-binding protein CP43 is an inner subunit of the Photosystem II (PSII) reaction center core complex of all oxygenic photoautotrophs. X-Ray structural evidence places the guanidinium cation of the conserved arginine 357 residue of CP43 within a few Angstroms to the Mn(4)Ca cluster of the water-oxidizing complex (WOC) and has been implicated as a possible carbonate binding site. To test the hypothesis, the serine mutant, CP43-R357S, from Synechocystis PCC 6803 was investigated by PSII variable fluorescence (F(v)/F(m)) and simultaneous flash O(2) yield measurements in cells and thylakoid membranes. The R357S mutant assembles PSII-WOC centers, but is unable to grow photoautotrophically. Reconstitution of O(2) evolution by photoactivation and the occurrence of period-four oscillations of F(v)/F(m) establishes that the R357S mutant contains an assembled Mn(4)Ca cluster, but turnover is impaired as seen by an 11-fold larger Kok double miss parameter and faster decay of upper S states. Using pulsed light to avoid photoinactivation, wild-type cells and thylakoid membranes exhibit a 2-4-fold loss in O(2) evolution rate upon partial bicarbonate depletion under multiple turnover conditions, while the R357S mutant is unaffected by bicarbonate. Arginine R357 appears to function in binding a (bi)carbonate ion essential to normal catalytic turnover of the WOC. The quantum yield of electron donation from the WOC into PSII increases with decreasing turnover rate in R357S mutant cells and involves an aborted two-flash pathway that is distinct from the classical four-flash pattern. We speculate that an altered photochemical mechanism for O(2) production occurs via formation of hydrogen peroxide, by analogy to other treatments that retard the kinetics of proton release into the lumen.

Alterations of the oxygen-evolving apparatus induced by a 305Arg --> 305Ser mutation in the CP43 protein of photosystem II from Synechocystis sp. PCC 6803 under chloride-limiting conditions.

The psbC gene encodes CP43, a component of Photosystem II (PSII) in higher plants, algae, and cyanobacteria. Previous work demonstrated that alteration of an arginine residue occurring at position 305 to serine produced a strain (R305S) with altered PSII activity (Knoepfle, N., Bricker, T. M., and Putnam-Evans, C. (1999) Biochemistry 38, 1582-1588). This strain grew at wild-type rates in complete BG-11 media (480 microM chloride) and evolved oxygen at rates that were 60-70% of the observed wild-type rates. The R305S strain assembled approximately 70-80% of the functional PSII centers contained in the control strain, and these PSII centers were very sensitive to photoinactivation at high light intensities. We recently observed that the R305S mutant exhibited a pronounced chloride effect. When this mutant was grown in media depleted of chloride (30 microM chloride), it exhibited a severely reduced photoautotrophic growth rate. 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. Oxygen evolution rates for the mutant were further depressed to about 22% of that observed in control cells under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. The mutant exhibited a significantly lower relative quantum yield for oxygen evolution than did the control strain, and this was exacerbated under chloride-limiting conditions. Fluorescence yield measurements indicated that both the mutant and the control strains assembled fewer PSII reaction centers under chloride-limiting conditions. The reaction centers assembled by the mutant exhibited an enhanced sensitivity to photoinactivation under chloride-limiting conditions, with a t(1/2) of photoinactivation of 2.6 min under chloride-limiting conditions as compared to a t(1/2) of 4.7 min under normal growth conditions. The mutant also exhibited an enhanced stability of its S(2) state and increased number of centers in the S(1) state following dark incubation. These results indicate that the mutant R305S exhibits a defect in its ability to utilize chloride in support of efficient oxygen evolution in PSII. This is the first mutant of this type described in the CP43 protein.

Introduction of the 305Arg-->305Ser mutation in the large extrinsic loop E of the CP43 protein of Synechocystis sp. PCC 6803 leads to the loss of cytochrome c(550) binding to Photosystem II.

CP43, a component of Photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbC gene. Previous work demonstrated that alteration of an arginine residue occurring at position 305 to serine produced a strain (R305S) with altered PSII characteristics including lower oxygen-evolving activity, fewer assembled reaction centers, higher sensitivity to photoinactivation, etc. [Biochemistry 38 (1999) 1582]. Additionally, it was determined that the mutant exhibited an enhanced stability of its S2 state. Recently, we observed a significant chloride effect under chloride-limiting conditions. The mutant essentially lost the ability to grow photoautotrophically, assembled fewer fully functional PSII reaction centers and exhibited a very low rate of oxygen evolution. Thus, the observed phenotype of this mutation is very similar to that observed for the Delta(psb)V mutant, which lacks cytochrome c550 (Biochemistry 37 (1998) 1551). A His-tagged version of the R305S mutant was produced to facilitate the isolation of PSII particles. These particles were analyzed for the presence of cytochrome c550. Reduced minus oxidized difference spectroscopy and chemiluminescence examination of Western blots indicated that cytochrome c550 was absent in these PSII particles. Whole cell extracts from the R305S mutant, however, contained a similar amount of cytochrome c550 to that observed in the control strain. These results indicate that the mutation R305S in CP43 prevents the strong association of cytochrome c550 with the PSII core complex. We hypothesize that this residue is involved in the formation of the binding domain for the cytochrome.

Posttranslational modifications in the CP43 subunit of photosystem II.

Photosystem II (PSII) catalyzes the light-driven oxidation of water and the reduction of plastoquinone; the oxidation of water occurs at a cluster of four manganese. The PSII CP43 subunit functions in light harvesting, and mutations in the fifth luminal loop (E) of CP43 have established its importance in PSII structure and/or assembly [Kuhn, M. G. & Vermaas, V. F. J. (1993) Plant Mol. Biol. 23, 123-133]. The sequence A(350)PWLEPLR(357) in luminal loop E is conserved in CP43 genes from 50 organisms. To map important posttranslational modifications in this sequence, tandem mass spectrometry (MS/MS) was used. These data show that the indole side chain of Trp-352 is posttranslationally modified to give mass shifts of +4, +16, and +18 daltons. The masses of the modifications suggest that the tryptophan is modified to kynurenine (+4), a keto-/amino-/hydroxy- (+16) derivative, and a dihydro-hydroxy- (+18) derivative of the indole side chain. Peptide synthesis and MS/MS confirmed the kynurenine assignment. The +16 and +18 tryptophan modifications may be intermediates formed during the oxidative cleavage of the indole ring to give kynurenine. The site-directed mutations, W352C, W352L, and W352A, exhibit an increased rate of photoinhibition relative to wild type. We hypothesize that Trp-352 oxidative modifications are a byproduct of PSII water-splitting or electron transfer reactions and that these modifications target PSII for turnover. As a step toward understanding the tertiary structure of this CP43 peptide, structural modeling was performed by using molecular dynamics.