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William A Cramer

Publications and source records attributed to William A Cramer.

At least 19 recordsLinked to original sources
PubMed2006

Heme-heme interactions in the cytochrome b6f complex: EPR spectroscopy and correlation with structure.

Cytochrome b6f of oxygenic photosynthesis was studied using multifrequency, multimode EPR Spectroscopy. Frequency dependent signals above g = 4.3, and the observation of parallel-mode signals, are indicative of spin interactions in the complex. We demonstrate the presence of an exchange interaction between the unique high-spin heme cn and a nearby low-spin heme bn, and show that a quinone analog NQNO binds at or near to heme cn. The two hemes remain spin coupled upon the binding of NQNO, though strength of interaction decreases significantly. The electronic coupling implies that the heme bn/cn pair could function as a unit to facilitate 2-electron reduction of plastoquionone without generation of an energetically unfavorable semiquinone intermediate.

Cysteine↗
PubMed2006

Minimum length requirement of the flexible N-terminal translocation subdomain of colicin E3.

The 315-residue N-terminal T domain of colicin E3 functions in translocation of the colicin across the outer membrane through its interaction with outer membrane proteins including the OmpF porin. The first 83 residues of the T domain are known from structure studies to be disordered. This flexible translocation subdomain contains the TolB box (residues 34 to 46) that must cross the outer membrane in an early translocation event, allowing the colicin to bind to the TolB protein in the periplasm. In the present study, it was found that cytotoxicity of the colicin requires a minimum length of 19 to 23 residues between the C terminus (residue 46) of the TolB box and the end of the flexible subdomain (residue 83). Colicin E3 molecules of sufficient length display normal binding to TolB and occlusion of OmpF channels in vitro. The length of the N-terminal subdomain is critical because it allows the TolB box to cross the outer membrane and interact with TolB. It is proposed that the length constraint is a consequence of ordered structure in the downstream segment of the T domain (residues 84 to 315) that prevents its insertion through the outer membrane via a translocation pore that includes OmpF.

Bacterial Outer Membrane Proteins↗
PubMed2006

The colicin E3 outer membrane translocon: immunity protein release allows interaction of the cytotoxic domain with OmpF porin.

The crystal structure previously obtained for the complex of BtuB and the receptor binding domain of colicin E3 forms a basis for further analysis of the mechanism of colicin import through the bacterial outer membrane. Together with genetic analysis and studies on colicin occlusion of OmpF channels, this implied a colicin translocon consisting of BtuB and OmpF that would transfer the C-terminal cytotoxic domain (C96) of colicin E3 through the Escherichia coli outer membrane. This model does not, however, explain how the colicin attains the unfolded conformation necessary for transfer. Such a conformation change would require removal of the immunity (Imm) protein, which is bound tightly in a complex with the folded colicin E3. In the present study, it was possible to obtain reversible removal of Imm in vitro in a single column chromatography step without colicin denaturation. This resulted in a mostly unordered secondary structure of the cytotoxic domain and a large decrease in stability, which was also found in the receptor binding domain. These structure changes were documented by near- and far-UV circular dichroism and intrinsic tryptophan fluorescence. Reconstitution of Imm in a complex with C96 or colicin E3 restored the native structure. C96 depleted of Imm, in contrast to the native complex with Imm, efficiently occluded OmpF channels, implying that the presence of tightly bound Imm prevents its unfolding and utilization of the OmpF porin for subsequent import of the cytotoxic domain.

Bacterial Outer Membrane Proteins↗
PubMed2006

Consequences of the structure of the cytochrome b6f complex for its charge transfer pathways.

At least two features of the crystal structures of the cytochrome b6f complex from the thermophilic cyanobacterium, Mastigocladus laminosus and a green alga, Chlamydomonas reinhardtii, have implications for the pathways and mechanism of charge (electron/proton) transfer in the complex: (i) The narrow 11 x 12 A portal between the p-side of the quinone exchange cavity and p-side plastoquinone/quinol binding niche, through which all Q/QH2 must pass, is smaller in the b6f than in the bc1 complex because of its partial occlusion by the phytyl chain of the one bound chlorophyll a molecule in the b6f complex. Thus, the pathway for trans-membrane passage of the lipophilic quinone is even more labyrinthine in the b6f than in the bc1 complex. (ii) A unique covalently bound heme, heme cn, in close proximity to the n-side b heme, is present in the b6f complex. The b6f structure implies that a Q cycle mechanism must be modified to include heme cn as an intermediate between heme bn and plastoquinone bound at a different site than in the bc1 complex. In addition, it is likely that the heme bn-cn couple participates in photosytem I-linked cyclic electron transport that requires ferredoxin and the ferredoxin: NADP+ reductase. This pathway through the n-side of the b6f complex could overlap with the n-side of the Q cycle pathway. Thus, either regulation is required at the level of the redox state of the hemes that would allow them to be shared by the two pathways, and/or the two different pathways are segregated in the membrane.

Amino Acid Sequence↗
PubMed2006

Sequencing covalent modifications of membrane proteins.

A number of strategies have successfully extended plant proteomics into the bilayer domain. Important benefits can be afforded by including a well-resolved intact protein mass spectrum alongside peptide identification experiments; recent studies of thylakoid membranes have yielded new information on the primary structure and covalent post-translational modification of many of the integral proteins. Intact mass proteomics is advancing through the development of core technologies in separations and mass spectrometry, with the goal of providing comprehensive primary structure coverage that includes transmembrane domains with reliable measurements of protein abundance and turnover. To address the limitations associated with separation technologies such as 2D-gel electrophoresis, alternative systems are being investigated and 2D-liquid chromatography of thylakoid membrane proteins, using both denaturing and non-denaturing first dimensions, has been successful, extending separation space and providing intact protein solutions for electrospray-ionization mass spectrometry and top-down proteomics. High-resolution conventional, and Fourier-transform, mass spectrometry is bringing increasing resolution to tandem mass spectrometry allowing for 'top-down' mass spectrometry of intact proteins. Thus the core chromatographic technologies already developed for intact mass proteomics of integral membrane proteins also allow their 'top-down' analysis. Thylakoid membrane proteins with one and two transmembrane helices have been analysed demonstrating the ability of collision-activated dissociation (CAD) to sequence through transmembrane domains. When analysed by top-down proteomics, the small subunits of the cytochrome b6f complex from Arabidopsis thylakoids reveal novel post-transcriptional/translational modifications including the presence of glutamic acid at position 2 of PetL instead of the proline residue predicted from the gene sequence.

Chromatography, Agarose↗
PubMed2006

Lipid dependence of the channel properties of a colicin E1-lipid toroidal pore.

Colicin E1 belongs to a group of bacteriocins whose cytotoxicity toward Escherichia coli is exerted through formation of ion channels that depolarize the cytoplasmic membrane. The lipid dependence of colicin single-channel conductance demonstrated intimate involvement of lipid in the structure of this channel. The colicin formed "small" conductance 60-picosiemens (pS) channels, with properties similar to those previously characterized, in 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (C20) or thinner membranes, whereas it formed a novel "large" conductance 600-pS state in thicker 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22) bilayers. Both channel states were anion-selective and voltage-gated and displayed a requirement for acidic pH. Lipids having negative spontaneous curvature inhibited the formation of both channels but increased the ratio of open 600 pS to 60 pS conductance states. Different diameters of small and large channels, 12 and 16 A, were determined from the dependence of single-channel conductance on the size of nonelectrolyte solute probes. Colicin-induced lipid "flip-flop" and the decrease in anion selectivity of the channel in the presence of negatively charged lipids implied a significant contribution of lipid to the structure of the channel, most readily described as toroidal organization of lipid and protein to form the channel pore.

Colicins↗
PubMed2006

Fe vibrational spectroscopy of myoglobin and cytochrome f.

The Fe vibrational density of states (VDOS) has been determined for the heme proteins deoxymyoglobin, metmyoglobin, and cytochrome f in the oxidized and reduced states, using nuclear resonance vibrational spectroscopy (NRVS). For cytochrome f in particular, the NRVS spectrum is compared with multiwavelength resonance Raman spectra to identify those Raman modes with significant Fe displacement. Modes not seen by Raman due to optical selection rules appear in the NRVS spectrum. The mean Fe force constant extracted from the VDOS illustrates how Fe dynamics varies among these four monoheme proteins, and is correlated with oxidation and spin state trends seen in model heme compounds. The protein's contribution to Fe motion is dominant at low frequencies, where coupling to the backbone tightly constrains Fe displacements in cytochrome f, in contrast to enhanced heme flexibility in myoglobin.

Cytochromes f↗
PubMed2006

Transmembrane traffic in the cytochrome b6f complex.

Crystal structures and their implications for function are described for the energy transducing hetero-oligomeric dimeric cytochrome b6f complex of oxygenic photosynthesis from the thermophilic cyanobacterium, Mastigocladus laminosus, and the green alga, Chlamydomonas reinhardtii. The complex has a cytochrome b core and a central quinone exchange cavity, defined by the two monomers that are very similar to those in the respiratory cytochrome bc1 complex. The pathway of quinol/quinone (Q/QH2) transfer emphasizes the labyrinthine internal structure of the complex, including an 11x12 A portal through which Q/QH2, containing a 45-carbon isoprenoid chain, must pass. Three prosthetic groups are present in the b6f complex that are not found in the related bc1 complex: a chlorophyll (Chl) a, a beta-carotene, and a structurally unique covalently bound heme that does not possess amino acid side chains as axial ligands. It is hypothesized that this heme, exposed to the cavity and a neighboring plastoquinone and close to the positive surface potential of the complex, can function in cyclic electron transport via anionic ferredoxin.

Algal Proteins↗
PubMed2005

Intraprotein transfer of the quinone analogue inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone in the cytochrome b6f complex.

Details are presented of the structural analysis of the cytochrome b(6)f complex from the thermophilic cyanobacterium, Mastigocladus laminosus, in the presence of the electrochemically positive (p)-side quinone analogue inhibitor, 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). One DBMIB binding site was found. This site is peripheral to the quinone binding space defined by the binding sites of other p-side inhibitors previously resolved in cytochrome bc(1)/b(6)f complexes. This high-affinity site resides in a p-side interfacial niche bounded by cytochrome f, subunit IV, and cytochrome b(6), is close (8 A) to the p-side heme b, but distant (19 A) from the [2Fe-2S] cluster. No significant electron density associated with the DBMIB was found elsewhere in the structure. However, the site at which DBMIB can inhibit light-induced redox turnover is within a few A of the [2Fe-2S] cluster, as shown by the absence of inhibition in mutants of Synechococcus sp. PCC 7002 at iron sulfur protein-Leu-111 near the cluster. The ability of a minimum amount of initially oxidized DBMIB to inhibit turnover of WT complex after a second light flash implies that there is a light-activated movement of DBMIB from the distal peripheral site to an inhibitory site proximal to the [2Fe-2S] cluster. Together with the necessary passage of quinone/quinol through the small Q(p) portal in the complex, it is seen that transmembrane traffic of quinone-like molecules through the core of cytochrome bc complexes can be labyrinthine.

Binding Sites↗
PubMed2005

The single chlorophyll a molecule in the cytochrome b6f complex: unusual optical properties protect the complex against singlet oxygen.

The cytochrome b(6)f complex of oxygenic photosynthesis mediates electron transfer between the reaction centers of photosystems I and II and facilitates coupled proton translocation across the membrane. High-resolution x-ray crystallographic structures (Kurisu et al., 2003; Stroebel et al., 2003) of the cytochrome b(6)f complex unambiguously show that a Chl a molecule is an intrinsic component of the cytochrome b(6)f complex. Although the functional role of this Chl a is presently unclear (Kuhlbrandt, 2003), an excited Chl a molecule is known to produce toxic singlet oxygen as the result of energy transfer from the excited triplet state of the Chl a to oxygen molecules. To prevent singlet oxygen formation in light-harvesting complexes, a carotenoid is typically positioned within approximately 4 A of the Chl a molecule, effectively quenching the triplet excited state of the Chl a. However, in the cytochrome b(6)f complex, the beta-carotene is too far (> or =14 Angstroms) from the Chl a for effective quenching of the Chl a triplet excited state. In this study, we propose that in this complex, the protection is at least partly realized through special arrangement of the local protein structure, which shortens the singlet excited state lifetime of the Chl a by a factor of 20-25 and thus significantly reduces the formation of the Chl a triplet state. Based on optical ultrafast absorption difference experiments and structure-based calculations, it is proposed that the Chl a singlet excited state lifetime is shortened due to electron exchange transfer with the nearby tyrosine residue. To our knowledge, this kind of protection mechanism against singlet oxygen has not yet been reported for any other chlorophyll-containing protein complex. It is also reported that the Chl a molecule in the cytochrome b(6)f complex does not change orientation in its excited state.

Biophysical Phenomena↗
PubMed2005

Problems in obtaining diffraction-quality crystals of hetero-oligomeric integral membrane proteins.

The major barrier responsible for the slow pace of structure determination of integral membrane proteins is the difficulty of crystallizing detergent-solubilized hydrophobic proteins, particularly hetero-oligomeric integral membrane proteins. For the latter class of multi-subunit proteins, we have encountered the following problems in addition to the ubiquitous problem of detergent compatibility: (i) instability caused by over-purification that results in delipidation; (ii) protease activity degrading exposed loops and termini of subunits of the complex that could not be inhibited; (iii) poor protein-protein contacts presumably arising from masking by the detergent micelle. Problem (i) could be ameliorated in crystallization of the cytochrome b(6)f complex by augmenting the delipidated complex with synthetic lipid. Problem (ii) has not been solved. Problem (iii) has been solved in other systems by the use of monoclonal antibodies (or other protein ligands) to increase the probability of protein-protein contacts. In the case of the complex formed by the cobalamin and colicin receptor, BtuB, and the receptor binding domain of colicin E3, the latter served as a ligand for protein-protein contacts that facilitated crystallization.

Crystallization↗
PubMed2005

Structure of the cytochrome b6f complex: new prosthetic groups, Q-space, and the 'hors d'oeuvres hypothesis' for assembly of the complex.

3-A crystal structures of the cytochrome b6f complex have provided a structural framework for the photosynthetic electron transport chain. The structures of the 220,000 molecular weight dimeric cytochrome b6f complex from the thermophilic cyanobacterium, Mastigocladis laminosus (Kurisu et al. 2003, Science 302: 1009-1014), and the green alga, Chlamydomonas reinhardtii (Stroebel et al. 2003, Nature 426: 413-418), are very similar. The latter is the first structure of a integral membrane photosynthetic electron transport complex from a eukaryotic source. The M. laminosus and C. reinhardtii structures have provided structural information and experimental insights to the properties and functions of three native and novel prosthetic groups, a chlorophyll a, a beta-carotene, and a unique heme x, one copy of which is found in each monomer of the cytochrome b6f complex, but not the cytochrome bc1 complex from the mitochondrial respiratory chain of animals and yeast. Several functional insights have emerged from the structures including the function of the dimer; the properties of heme x; the function of the inter-monomer quinone-exchange cavity; a quinone diffusion pathway through relatively narrow crevices or portals; a modified reaction scheme for n-side quinone redox reactions; a necessarily novel mechanism for quenching of the bound chlorophyll triplet state; a possible role for the bound chlorophyll a in activation of the LHC kinase; and a structural and assembly role for the four small PetG, L, M, and N subunits. An 'hors d'oeuvres hypothesis' for assembly of the complex is proposed for the small 'hydrophobic stick' or 'picket fence' polypeptides at the periphery of the complex, based on the cis-positive orientation of the small hydrophobic subunits and the 'toothpick' binding mode of the beta-carotene.

Animals↗
PubMed2004

Characterization of the high-spin heme x in the cytochrome b6f complex of oxygenic photosynthesis.

X-ray structures at 3.0-3.1 A resolution of the cytochrome b(6) f complex from the cyanobacterium Mastigocladus laminosus [Kurisu, G., Zhang, H., Smith, J. L., and Cramer, W. A. (2003) Science 302, 1009-1014] and the green alga Chlamydomonas reinhardtii [Stroebel, D., Choquet, Y., Popot, J.-L., and Picot, D. (2003) Nature 426, 413-418] showed the presence of a unique heme, hemex, that is covalently linked by a single thioether bond to a Cys residue (Cys35) on the electrochemically negative (n) side of the cytochrome b(6) polypeptide. Heme x faces the intermonomer quinone exchange cavity. The only axial ligand associated with this heme is a H(2)O or OH(-) that is H-bonded to the propionate of the stromal side heme b(n), showing that it is pentacoordinate. The spectral properties of this heme were hardly defined at the time of the structure determination. The pyridine hemochromagen redox difference spectrum for heme x covalently bound to the cytochrome b polypeptide isolated from SDS-PAGE displays a low-amplitude broad spectrum with a peak at 553 nm, similar to that of other hemes with a single thioether linkage. The binding of CO and a hydrophobic cyanide analogue, butyl isocyanide, to dithionite-reduced b(6) f complex perturbs and significantly shifts the redox difference visible spectrum. Together with EPR spectra displaying g values of the oxidized complex of 6.7 and 7.4, heme x is defined as a ferric high-spin heme in a rhombic environment. In addition to a possible function in photosystem I-linked cyclic electron transport, the five-coordinate state implies that there is at least one more function of heme x that is related to axial binding of a physiological ligand.

Binding Sites↗
PubMed2004

Molecular control of a bimodal distribution of quinone-analogue inhibitor binding sites in the cytochrome b(6)f complex.

The 3.0-3.1A X-ray structures of the cytochrome b(6)f complex from Mastigocladus laminosus and Chlamydomonas reinhardtii obtained in the presence of the p-side quinone-analogue inhibitor tridecyl-stigmatellin (TDS) are very similar. A difference occurs in the p-side binding position of TDS. In C.reinhardtii, TDS binds in the ring-in mode, as previously found for stigmatellin in X-ray structures of the cytochrome bc(1) complex. In this mode, the H-bonding chromone ring moiety of the TDS bound in the Q(p) niche is proximal to the ISP [2Fe-2S] cluster, and its 13 carbon tail extends through a portal to the large inter-monomer quinone-exchange cavity. However, in M.laminosus, TDS binds in an oppositely oriented ring-out mode, with the tail inserted toward the Q(p) niche through the portal and the ring caught in the quinone-exchange cavity that is 20A away from the [2Fe-2S] cluster. Site-directed mutagenesis of residues that might determine TDS binding was performed with the related transformable cyanobacterium Synechococcus sp. PCC 7002. The following changes in the sensitivity of electron transport activity to TDS and stigmatellin were observed: (a) little effect of mutation L193A in cytochrome b(6), which is proximal to the chromone of the ring-out TDS; (b) almost complete loss of sensitivity by mutation L111A in the ISP cluster binding region, which is close to the chromone of the ring-in TDS; (c) a ten and 60-fold increase associated with the mutation L81F in subunit IV. It was inferred that only the ring-in binding mode, in which the ring interacts with residues near the ISP, is inhibitory, and that residue 81 of subunit IV, which resides at the immediate entrance to the Q(p) niche, controls the relative binding affinity of inhibitor at the two different binding sites.

Amino Acid Sequence↗
PubMed2004

On the role of lipid in colicin pore formation.

Insights into the protein-membrane interactions by which the C-terminal pore-forming domain of colicins inserts into membranes and forms voltage-gated channels, and the nature of the colicin channel, are provided by data on: (i) the flexible helix-elongated state of the colicin pore-forming domain in the fluid anionic membrane interfacial layer, the optimum anionic surface charge for channel formation, and voltage-gated translocation of charged regions of the colicin domain across the membrane; (ii) structure-function data on the voltage-gated K(+) channel showing translocation of an arginine-rich helical segment through the membrane; (iii) toroidal channels formed by small peptides that involve local participation of anionic lipids in an inverted phase. It is proposed that translocation of the colicin across the membrane occurs through minimization of the Born charging energy for translocation of positively charged basic residues across the lipid bilayer by neutralization with anionic lipid head groups. The resulting pore structure may consist of somewhat short, ca. 16 residues, trans-membrane helices, in a locally thinned membrane, together with surface elements of inverted phase lipid micelles.

Colicins↗
PubMed2004

Effect of lipids with different spontaneous curvature on the channel activity of colicin E1: evidence in favor of a toroidal pore.

The channel activity of colicin E1 was studied in planar lipid bilayers and liposomes. Colicin E1 pore-forming activity was found to depend on the curvature of the lipid bilayer, as judged by the effect on channel activity of curvature-modulating agents. In particular, the colicin-induced trans-membrane current was augmented by lysophosphatidylcholine and reduced by oleic acid, agents promoting positive and negative membrane curvature, respectively. The data obtained imply direct involvement of lipids in the formation of colicin E1-induced pore walls. It is inferred that the toroidal pore model previously validated for small antimicrobial peptides is applicable to colicin E1, a large protein that contains ten alpha-helices in its pore-forming domain.

Amino Acid Sequence↗