AHl2 is not sufficient to pump two protons.6 This observation contradicts those reports indicating that two protons are pumped per photocycle. However, our results are consistent with those experimental studies that indicate that the number of protons pumped per photocycle is approximately equal to -O .~/ @ J~ (see discussion in refs 5 and 16-19).In closing, we note that the revised assignments for 0' and a2 affect not only AHl2 but also the value of xK (the fraction of the K photoproduct in the photostationary state) and the calculated spectrum of K. Our previous value of xK (A = 500 nm) of 0.46 increases to 0.53 (Table 11, ref 6) on the basis of = 0.68, which is equal within experimental error to the 510-nm value of 0.56 reported in ref 14. A recalculated K spectrum based on our bR spectrum and our K -bR difference spectrum assuming = 0.68 is nearly identical with that shown in ref 14.This means that our raw spectroscopic data are consistent with those presented in ref 14, and this observation supports the use of our raw spectroscopic data to assign the partition functions that appear in eqs 1-3 as a function of and aZ. The fact that our photocalorimetry data are more consistent with a ratio of = 0.45 versus the revised value of @'/a2 = 0.68 is reflected in our least-squares regression by a -1 .S-fold percentage increase in the standard deviation for our revised enthalpy (1 1.6 i 3.4 (29%) kcal mol-') relative to the standard deviation associated with our previous assignment (15.9 f 3.2 (20%) kcal mol-').

CopZ, an Atx1-like copper chaperone from the bacterium Bacillus subtilis, functions as part of a complex cellular machinery for Cu(I) trafficking and detoxification, in which it interacts specifically with the transmembrane Cu(I)-transporter CopA. Here we demonstrate that the cysteine residues of the MXCXXC Cu(I)-binding motif of CopZ have low proton affinities, with both exhibiting pK(a) values of 6 or below. Chelator competition experiments demonstrated that the protein binds Cu(I) with extremely high affinity, with a small but significant pH-dependence over the range pH 6.5-8.0. From these data, a pH-corrected formation constant, beta(2)= approximately 6 x 10(22) M(-2), was determined. Rapid exchange of Cu(I) between CopZ and the Cu(I)-chelator BCS (bathocuproine disulfonate) indicated that the mechanism of exchange does not involve simple dissociation of Cu(I) from CopZ (or BCS), but instead proceeds via the formation of a transient Cu(I)-mediated protein-chelator complex. Such a mechanism has similarities to the Cu(I)-exchange pathway that occurs between components of copper-trafficking pathways.

No abstract

The establishment of symbiotic associations in plants requires calcium oscillations that must be decoded to invoke downstream developmental programs. In animal systems, comparable calcium oscillations are decoded by calmodulin (CaM)–dependent protein kinases, but symbiotic signaling involves a calcium/CaM–dependent protein kinase (CCaMK) that is unique to plants. CCaMK differs from the animal CaM kinases by its dual ability to bind free calcium, via calcium binding EF-hand domains on the protein, or to bind calcium complexed with CaM, via a CaM binding domain. In this study, we dissect this dual regulation of CCaMK by calcium. We find that calcium binding to the EF-hand domains promotes autophosphorylation, which negatively regulates CCaMK by stabilizing the inactive state of the protein. By contrast, calcium-dependent CaM binding overrides the effects of autophosphorylation and activates the protein. The differential calcium binding affinities of the EF-hand domains compared with those of CaM suggest that CCaMK is maintained in the inactive state at basal calcium concentrations and is activated via CaM binding during calcium oscillations. This work provides a model for decoding calcium oscillations that uses differential calcium binding affinities to create a robust molecular switch that is responsive to calcium concentrations associated with both the basal state and with oscillations.

Conformations of (R)-alpha-(N6-adenyl)styrene oxide adducts at positions X6 in d(CGGACXAGAAG).d(CTTCTTGTCCG) and X7 in d(CGGACAXGAAG).d(CTTCTTGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human n-ras protooncogene, were refined from 1H NMR data. These were the R(61,2) and R(61,3) adducts. Chemical shift perturbations were in the 5'-direction from the sites of adduction; large changes were observed for C5 H5 and H6 in the R(61,2) adduct. The styrene moieties were only partially defined by NOE data. Spectral overlap, particularly for the R(61,2) adduct, prevented complete assignments of the aromatic resonances; likewise, there were insufficient data to orient the CH2OH moieties. Ring flips were slow on the NMR time scale. For the R(61,2) adduct 260 restraints were obtained from NOE data at three mixing times using relaxation matrix analysis; for the R(61,3) adduct 230 restraints were obtained. Structures emergent from molecular dynamics/simulated annealing for the R(61,2) adduct converged to average and maximum pairwise rms differences of 1.3 and 1.7 A, respectively, while those for the R(61,3) adduct converged to average and maximum pairwise rms differences of 1.2 and 1.6 A. Sixth root residual indices of 7.5 and 6.8 x 10(-2) were measured between the refined structures and NOE intensities using relaxation matrix calculations for the R(61,2) and R(61,3) adducts, respectively. The styrene rings were in the 5'-direction from the lesion sites in the major groove. The preferred orientation calculated for the R(61,2) adduct placed the styrene ring edgewise and approximately orthogonal to C5, while that calculated for the R(61,3) duplex had the styrene ring approximately orthogonal to the major groove edges of base pairs A6.T17 and R-SOA7.T16.

The (S)-alpha-(N6-adenyl)styrene oxide adducts at positions X6 in d(CGGACXAGAAG). d(CTTCTTGTCCG) and X7 in d(CGGACAXGAAG).d(CTTCTTGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human n-ras protooncogene, were examined by 1H NMR. These were the S(61,2) and S(61,3) adducts. Chemical shift perturbations were in the 3'-direction from the sites of adduction; upfield changes associated with the styrene aromatic ring current were noted for S-SOA6 H2 and H1', T16 N3H, H6, and CH3 resonances in the S(61,2) oligomer. In the S(61,3) oligomer, S-SOA7 H1', T16 H1', C15 N4Ha, and H5 shifted upfield. The styrene aromatic rings flipped rapidly on the NMR time scale; under these conditions the ortho and meta aromatic protons were equivalent. A sequence effect, in which the S(61,2) adduct equilibrated between two conformers, while the S(61,3) adduct exhibited only a single conformation, was observed. Potential energy minimization of the S(61,2) adduct major conformation yielded a structure in which the styrene ring was oriented in the 3'-direction and interacted primarily with the complementary strand. For the S(61,3) adduct, 291 restraints were obtained from NOE data at three mixing times using relaxation matrix analysis. The emergent structures refined to an average rms difference of 1.3 A, determined by pairwise analysis. These were compared to NOE intensity data; the calculated sixth root residual index was 9.2 x 10-2 at 250 ms. In the refined structure, the styrene ring was also oriented in the 3'-direction and interacted with the complementary strand. The minor conformation of the S(61,2) adduct was not identified. These results contrasted with the diastereomeric R(61,2) and R(61,3) adducts, which underwent slow ring flips on the NMR time scale and for which small sequence effects involving the minimum energy conformation of the styrene ring were observed.

Copper trafficking proteins and copper-sensitive regulators are often found to be able to bind multiple Cu(I) ions in the form of Cu(I) clusters. We have determined the high-resolution X-ray crystal structure of an Atx1-like copper chaperone protein from Bacillus subtilis containing a novel tetranuclear Cu(I) cluster. The identities and oxidation states of the cluster ions were established unambiguously by refinement of X-ray energy-dependent anomalous scattering factors. The [Cu(4)(S-Cys)(4)(N-His)(2)] cluster geometry provides new structural insights into not only the binding of multiple cuprous ions by metallochaperones but also protein-associated tetranuclear Cu(I) clusters, including those found in eukaryotic copper-responsive transcription factors.

CopA, a P‐type ATPase transporter involved in copper detoxification in Bacillus subtilis, contains two soluble Atx1‐like domains separated by a short linker at its N‐terminus, an arrangement that occurs widely in copper transporters from both prokaryotes and eukaryotes. Both domains were previously found to bind Cu(I) with very high affinity. Above a level of 1 Cu(I) per CopAab, dimerization occurred, leading to a highly luminescent multinuclear Cu(I) species [Singleton C & Le Brun NE (2009) Dalton Trans, 688–696]. To try to understand the contributions of each domain to the complex Cu(I)‐binding behaviour of this and related proteins, we purified a wild‐type form of the first domain (CopAa). In isolation, the domain bound Cu(I) with very high affinity (K = ∼ 1 × 1018 m−1) and underwent Cu(I)‐mediated protein association, resulting in a mixture of dimer and tetramer species. Addition of further Cu(I) up to 1 Cu(I) per CopAa monomer led to a weakly luminescent species, whereas further additions [2 Cu(I) per CopAa monomer] resulted in protein unfolding. Analysis of the MTCAAC binding motif Cys residue acid–base properties revealed pKa values of 5.7 and 7.3, consistent with the pH dependence of Cu(I) binding, and with the proposal that low proton affinity is associated with high Cu(I) affinity. Finally, Cu(I) exchange between CopAa and the chelator bathocuproine sulfonate revealed rapid exchange in both directions, demonstrating an interaction between the protein and the chelator that catalyses metal ion transfer. Overall, CopAa exhibits similarities to CopAab in terms of affinity and complexity of Cu(I) binding, but the details of Cu(I) binding are distinct. Structured digital abstract  http://www.uniprot.org/uniprot/o32220 and http://www.uniprot.org/uniprot/o32220 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8288724)  http://www.uniprot.org/uniprot/o32220 and http://www.uniprot.org/uniprot/o32220 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0028 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8288724)