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William A Cramer

Publications and source records attributed to William A Cramer.

34 records · Page 2Linked to original sources
PubMed2004

Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import.

The interaction of colicins with target cells is a paradigm for protein import. To enter cells, bactericidal colicins parasitize Escherichia coli outer membrane receptors whose physiological purpose is the import of essential metabolites. Colicins E1 and E3 initially bind to the BtuB receptor, whose beta-barrel pore is occluded by an N-terminal globular "plug". The x-ray structure of a complex of BtuB with the coiled-coil BtuB-binding domain of colicin E3 did not reveal displacement of the BtuB plug that would allow passage of the colicin (Kurisu, G., S. D. Zakharov, M. V. Zhalnina, S. Bano, V. Y. Eroukova, T. I. Rokitskaya, Y. N. Antonenko, M. C. Wiener, and W. A. Cramer. 2003. Nat. Struct. Biol. 10:948-954). This correlates with the inability of BtuB to form ion channels in planar bilayers, shown in this work, suggesting that an additional outer membrane protein(s) is required for colicin import across the outer membrane. The identity and interaction properties of this OMP were analyzed in planar bilayer experiments.OmpF and TolC channels in planar bilayers were occluded by colicins E3 and E1, respectively, from the trans-side of the membrane. Occlusion was dependent upon a cis-negative transmembrane potential. A positive potential reversibly opened OmpF and TolC channels. Colicin N, which uses only OmpF for entry, occludes OmpF in planar bilayers with the same orientation constraints as colicins E1 and E3. The OmpF recognition sites of colicins E3 and N, and the TolC recognition site of colicin E1, were found to reside in the N-terminal translocation domains. These data are considered in the context of a two-receptor translocon model for colicin entry into cells.

Bacterial Outer Membrane Proteins↗
PubMed2004

Evolution of photosynthesis: time-independent structure of the cytochrome b6f complex.

Structures of the cytochrome b(6)f complex obtained from the thermophilic cyanobacterium Mastigocladus laminosus and the green alga Chlamydomonas reinhardtii, whose appearance in evolution is separated by 10(9) years, are almost identical. Two monomers with a molecular weight of 110,000, containing eight subunits and seven natural prosthetic groups, are separated by a large lipid-containing "quinone exchange cavity". A unique heme, heme x, that is five-coordinated and high-spin, with no strong field ligand, occupies a position close to intramembrane heme b(n). This position is filled by the n-side bound quinone, Q(n), in the cytochrome bc(1) complex of the mitochondrial respiratory chain. The structure and position of heme x suggest that it could function in ferredoxin-dependent cyclic electron transport as well as being an intermediate in a quinone cycle mechanism for electron and proton transfer. The significant differences between the cyanobacterial and algal structures are as follows. (i) On the n-side, a plastoquinone molecule is present in the quinone exchange cavity in the cyanobacterial complex, and a sulfolipid is bound in the algal complex at a position corresponding to a synthetic DOPC lipid molecule in the cyanobacterial complex. (ii) On the p-side, in both complexes a quinone analogue inhibitor, TDS, passes through a portal that separates the large cavity from a niche containing the Fe(2)S(2) cluster. However, in the cyanobacterial complex, TDS is in an orientation that is the opposite of its position in the algal structure and bc(1) complexes, so its headgroup in the M. laminosus structure is 20 A from the Fe(2)S(2) cluster.

Algal Proteins↗
PubMed2004

On the mechanism and pathway of colicin import across the E. Coli outer membrane.

Colicins and phages parasitize outer membrane receptors whose physiological purpose is the transport of metabolites, metals, vitamins, and sugars. From mutagenesis studies, it is known that several colicins require the function of two outer membrane protein (Omp) receptors for cytotoxicity. A formidable list of problems associated with an understanding of a two receptor mechanism for colicin translocation includes the definition of the sites of initial binding and interactions of the colicin with the OM translocator protein, the working lumenal aperture of the translocator, the question of whether the colicin must be unfolded for translocation, the source of energy for unfolding and translocation, the order of colicin translocation, and the sites and mechanism of interaction of the colicins with the Tol-Pal proteins on the periplasmic side of the outer membrane. 3D crystal structures recently obtained of the cobalamin (vitamin B12) receptor (BtuB), and of the complex of BtuB with the 135 residue receptor binding domain (R135) of colicin E3, have provided some new insights on the interactions between two Omp receptors that are necessary for translocation of colicins. Together with spectroscopic data on the R135-BtuB interaction and electrophysiological data on the colicin E3-OmpF interaction, this has led to a proposal for the utilization of two receptors, BtuB-OmpF, in an outer membrane translocon for colicin E3.

Bacterial Outer Membrane Proteins↗
PubMed2004

Cytochrome bc complexes: a common core of structure and function surrounded by diversity in the outlying provinces.

The atomic-level picture of transmembrane protein complexes in the photosynthetic membrane has now been completed by the recent publication of crystal structures of cytochrome b(6)f and photosystem II. The two structures of cytochrome b(6)f, together with previously reported structures of the cytochrome bc(1) respiratory complex, provide a basis for understanding the central electron and proton transfer events of photosynthesis and respiration. The protein structures and charge transfer events within the core of the complexes are highly similar, but the complexes differ in subunit and chromophore composition in proportion to the distance from the central redox site within the membrane near the electropositive side.

Biological Transport↗
PubMed2004

Membrane protein crystallization in lipidic mesophases with tailored bilayers.

Monoacylglycerols have been used as bilayered hosts for growing crystals of membrane proteins. To date, the lipids used have had chains 16 and 18 carbon atoms long. We hypothesized that a shorter-chained lipid producing a thinner bilayer would facilitate the so-called in meso crystallization process. A 14 carbon monoacylglycerol was chosen as the lipid with which to test the proposal. To be compatible with the in meso method, a cis olefinic bond was placed in its acyl chain at a location arrived at by rational design. The target lipid was synthesized and was shown to form the requisite mesophase at room temperature. In support of the hypothesis, it produced crystals of bacteriorhodopsin and the outer membrane transporter, BtuB. The latter is the first beta barrel protein to be crystallized by the in meso method. Protein stability in the short-chain lipid and how this relates to crystallogenesis are discussed.

Chemistry Techniques, Analytical↗
PubMed2004

Ironies in photosynthetic electron transport: a personal perspective.

In this brief personal perspective, studies, mainly in my laboratory, directed toward understanding of the structure and function of the Fe-metalloprotein electron transport carriers of oxygenic photosynthesis are discussed. This level of understanding and the description of the events that led to this level are inevitably incomplete. The Fe-proteins considered are those, cytochromes b-559 (560), f, b (6), and x, and the Rieske [2Fe-2S] iron-sulfur protein, which have now been placed in a structural context as a result of X-ray diffraction analysis of crystals of Photosystem II and the cytochrome b (6) f complex and the Photosystem II reaction center.

Journal Article↗
PubMed2004

Purification and crystallization of the cytochrome b6f complex in oxygenic photosynthesis.

The cytochrome b6f complex from the thermophilic cyanobacterium Mastigocladus laminosus and spinach chloroplasts has been purified as a dimeric species. It was found by electrospray ionization mass spectroscopy to contain eight and nine subunits, respectively, and dimeric masses of 217,070 and 286,454 Da. The subunits common to the complex from both sources are petA (cytochrome f), B (cytochrome b6), C (Rieske iron-sulfur protein), D (subunit IV), and small 3.2-4.2 kDa polypeptides petG,L,M, and N. The ninth polypeptide, the 35 kDa petH poly-peptide in the spinach complex, was identified as ferredoxin NADP reductase (FNR), which binds to the complex tightly at a stoichiometry of approx 0.9 (cyt f)-1. The spinach complex contains diaphorase activity diagnostic of FNR, and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the cytochrome b6f complex. The purified cytochrome b6f complex contains stoichiometrically bound chlorophyll a and beta-carotene at a ratio of one per cytochrome f, and bound lipid, in which MGDG and PG are the most abundant species. The delipidated highly purified complexes are active immediately after preparation and for approx 1 wk if left on ice, transferring 300-350 electrons/cyt f/s. Both complexes are subject to proteolysis and associated loss of activity if left for extended periods (>1 wk) at room temperature. Addition of pure synthetic lipid to the delipidated M. laminosus complex (the "lipid augmentation" technique) allows rapid and ready formation of large (>0.2 mm) crystals suitable for x-ray diffraction analysis and structure determination, which diffract with good statistics to 3.0 A.

Bacterial Proteins↗
PubMed2003

The structure of BtuB with bound colicin E3 R-domain implies a translocon.

Cellular import of colicin E3 is initiated by the Escherichia coli outer membrane cobalamin transporter, BtuB. The 135-residue 100-A coiled-coil receptor-binding domain (R135) of colicin E3 forms a 1:1 complex with BtuB whose structure at a resolution of 2.75 A is reported. Binding of R135 to the BtuB extracellular surface (DeltaG(o) = -12 kcal mol(-1)) is mediated by 27 residues of R135 near the coiled-coil apex. Formation of the R135-BtuB complex results in unfolding of R135 N- and C-terminal ends, inferred to be important for unfolding of the colicin T-domain. Small conformational changes occur in the BtuB cork and barrel domains but are insufficient to form a translocation channel. The absence of a channel and the peripheral binding of R135 imply that BtuB serves to bind the colicin, and that the coiled-coil delivers the colicin to a neighboring outer membrane protein for translocation, thus forming a colicin translocon. The translocator was concluded to be OmpF from the occlusion of OmpF channels by colicin E3.

Bacterial Outer Membrane Proteins↗
PubMed2003

Structure of the cytochrome b6f complex of oxygenic photosynthesis: tuning the cavity.

The cytochrome b6f complex provides the electronic connection between the photosystem I and photosystem II reaction centers of oxygenic photosynthesis and generates a transmembrane electrochemical proton gradient for adenosine triphosphate synthesis. A 3.0 angstrom crystal structure of the dimeric b6f complex from the thermophilic cyanobacterium Mastigocladus laminosus reveals a large quinone exchange cavity, stabilized by lipid, in which plastoquinone, a quinone-analog inhibitor, and a novel heme are bound. The core of the b6f complex is similar to the analogous respiratory cytochrome bc1 complex, but the domain arrangement outside the core and the complement of prosthetic groups are strikingly different. The motion of the Rieske iron-sulfur protein extrinsic domain, essential for electron transfer, must also be different in the b6f complex.

Cell Membrane↗
PubMed2003

A defined protein-detergent-lipid complex for crystallization of integral membrane proteins: The cytochrome b6f complex of oxygenic photosynthesis.

The paucity of integral membrane protein structures creates a major bioinformatics gap, whose origin is the difficulty of crystallizing these detergent-solubilized proteins. The problem is particularly formidable for hetero-oligomeric integral membrane proteins, where crystallization is impeded by the heterogeneity and instability of the protein subunits and the small lateral pressure imposed by the detergent micelle envelope that surrounds the hydrophobic domain. In studies of the hetero (eight subunit)-dimeric 220,000 molecular weight cytochrome b(6)f complex, derived from the thermophilic cyanobacterium, Mastigocladus laminosus, crystals of the complex in an intact state could not be obtained from highly purified delipidated complex despite exhaustive screening. Crystals of proteolyzed complex could be obtained that grew very slowly and diffracted poorly. Addition to the purified lipid-depleted complex of a small amount of synthetic nonnative lipid, dioleolyl-phosphatidylcholine, resulted in a dramatic improvement in crystallization efficiency. Large crystals of the intact complex grew overnight, whose diffraction parameters are as follows: 94% complete at 3.40 A spacing; R(merge) = 8.8% (38.5%), space group, P6(1)22; and unit cell parameters, a = b = 156.3 A, c = 364.0 A, alpha = beta = 90 degrees, gamma = 120 degrees. It is proposed that the methodology of augmentation of a well-defined lipid-depleted integral membrane protein complex with synthetic nonnative lipid, which can provide conformational stability to the protein complex, may be of general use in the crystallization of integral membrane proteins.

Crystallization↗
PubMed2003

Functional insensitivity of the cytochrome b6f complex to structure changes in the hinge region of the Rieske iron-sulfur protein.

Structure analysis of the cytochrome bc1 complex in the presence and absence of Qp quinol analog inhibitors implied that a large amplitude motion of the Rieske iron-sulfur protein (ISP) is required to mediate electron transfer from ubiquinol to cytochrome c1. Studies of the functional consequences of mutagenesis of an 8-residue ISP "hinge" region in the bc1 complex showed it to be sensitive to structure perturbation, implying that optimum flexibility and length are required for the large amplitude motion. Mutagenesis-function analysis carried out on the ISP hinge region of the cytochrome b6 f complex using the cyanobacterium Synechococcus sp. PCC 7002 showed the following. (i) Of three petC genes, only that in the petCA operon codes for functional ISP. (ii) The function of the complex was insensitive to changes in the hinge region that increased flexibility, decreased flexibility by substitutions of 4-6 Pro residues, shortened the hinge by a 1-residue deletion, or elongated it by insertion of 4 residues. The latter change increased sensitivity to Qp inhibitors, whereas deletion of 2 residues resulted in a loss of inhibitor sensitivity and a decrease in activity, indicating a minimum hinge length of 7 residues required for optimum binding of ISP at the Qp site. Thus, in contrast to the bc1 complex, the function of the b6 f complex was insensitive to sequence changes in the ISP hinge that altered its length or flexibility. This implies that either the barriers to motion or the amplitude of ISP motion required for function is smaller than in the bc1 complex.

Amino Acid Sequence↗
PubMed2002

Colicin crystal structures: pathways and mechanisms for colicin insertion into membranes.

The X-ray structures of the channel-forming colicins Ia and N, and endoribonucleolytic colicin E3, as well as of the channel domains of colicins A and E1, and spectroscopic and calorimetric data for intact colicin E1, are discussed in the context of the mechanisms and pathways by which colicins are imported into cells. The extensive helical coiled-coil in the R domain and internal hydrophobic hairpin in the C domain are important features relevant to colicin import and channel formation. The concept of outer membrane translocation mediated by two receptors, one mainly used for initial binding and second for translocation, such as BtuB and TolC, respectively, is discussed. Helix elongation and conformational flexibility are prerequisites for import of soluble toxin-like proteins into membranes. Helix elongation contradicts suggestions that the colicin import involves a molten globule intermediate. The nature of the open-channel structure is discussed.

Bacterial Outer Membrane Proteins↗
PubMed2002

Electron transfer from the Rieske iron-sulfur protein (ISP) to cytochrome f in vitro. Is a guided trajectory of the ISP necessary for competent docking?

The time course of electron transfer in vitro between soluble domains of the Rieske iron-sulfur protein (ISP) and cytochrome f subunits of the cytochrome b(6)f complex of oxygenic photosynthesis was measured by stopped-flow mixing. The domains were derived from Chlamydomonas reinhardtii and expressed in Escherichia coli. The expressed 142-residue soluble ISP apoprotein was reconstituted with the [2Fe-2S] cluster. The second-order rate constant, k(2)((ISP-f)) = 1.5 x 10(6) m(-1) s(-1), for ISP to cytochrome f electron transfer was <10(-2) of the rate constant at low ionic strength, k(2)((f-PC))(> 200 x 10(6) m(-1) s(-1)), for the reduction of plastocyanin by cytochrome f, and approximately 1/30 of k(2)((f-PC)) at the ionic strength estimated for the thylakoid interior. In contrast to k(2)((f-PC)), k(2)((ISP-f)) was independent of pH and ionic strength, implying no significant role of electrostatic interactions. Effective pK values of 6.2 and 8.3, respectively, of oxidized and reduced ISP were derived from the pH dependence of the amplitude of cytochrome f reduction. The first-order rate constant, k(1)((ISP-f)), predicted from k(2)((ISP-f)) is approximately 10 and approximately 150 times smaller than the millisecond and microsecond phases of cytochrome f reduction observed in vivo. It is proposed that in the absence of electrostatic guidance, a productive docking geometry for fast electron transfer is imposed by the guided trajectory of the ISP extrinsic domain. The requirement of a specific electrically neutral docking configuration for ISP electron transfer is consistent with structure data for the related cytochrome bc(1) complex.

Cytochromes↗
PubMed2002

Tuning the membrane surface potential for efficient toxin import.

Membrane surface electrostatic interactions impose structural constraints on imported proteins. An unprecedented sensitive dependence on these constraints was seen in the voltage-gated import and channel formation by the C-terminal pore-forming domain of the bacteriocin, colicin E1. At physiological ionic strengths, significant channel current was observed only in a narrow interval of anionic lipid content ([L-]), with the maximum current (I(max)) at 25-30 mol% (dioleoyl)-phosphatidylglycerol ([L-]max) corresponding to a surface potential of the lipid bilayer in the absence of protein, psi(o)max = -60 +/- 5 mV. Higher ionic strength shifted [L-]max to larger values, but psi(o)max remained approximately constant. It is proposed that the channel current (i) increases and (ii) decreases at /psi(o)/ values <55 mV and >65 mV, because of (i) electrostatic interactions needed for effective insertion of the channel polypeptide and (ii) constraints due to electrostatic forces on the flexibility needed for cooperative insertion into the membrane. The loss of flexibility for /psi(o)/ 65 mV was demonstrated by the absence of thermally induced intraprotein distance changes of the bound polypeptide. The anionic lipid content, 25-30 mol%, corresponding to the channel current maxima, is similar to that of the target Escherichia coli cytoplasmic membrane and membranes of mesophilic microorganisms. This suggests that one reason the membrane surface potential is tuned in vivo is to facilitate protein import.

Anions↗
PubMed2002

Full subunit coverage liquid chromatography electrospray ionization mass spectrometry (LCMS+) of an oligomeric membrane protein: cytochrome b(6)f complex from spinach and the cyanobacterium Mastigocladus laminosus.

Highly active cytochrome b(6)f complexes from spinach and the cyanobacterium Mastigocladus laminosus have been analyzed by liquid chromatography with electrospray ionization mass spectrometry (LCMS+). Both size-exclusion and reverse-phase separations were used to separate protein subunits allowing measurement of their molecular masses to an accuracy exceeding 0.01% (+/-3 Da at 30,000 Da). The products of petA, petB, petC, petD, petG, petL, petM, and petN were detected in complexes from both spinach and M. laminosus, while the spinach complex also contained ferredoxin-NADP(+) oxidoreductase (Zhang, H., Whitelegge, J. P., and Cramer, W. A. (2001) Flavonucleotide:ferredoxin reductase is a subunit of the plant cytochrome b(6)f complex. J. Biol. Chem. 276, 38159-38165). While the measured masses of PetC and PetD (18935.8 and 17311.8 Da, respectively) from spinach are consistent with the published primary structure, the measured masses of cytochrome f (31934.7 Da, PetA) and cytochrome b (24886.9 Da, PetB) modestly deviate from values calculated based upon genomic sequence and known post-translational modifications. The low molecular weight protein subunits have been sequenced using tandem mass spectrometry (MSMS) without prior cleavage. Sequences derived from the MSMS spectra of these intact membrane proteins in the range of 3.2-4.2 kDa were compared with translations of genomic DNA sequence where available. Products of the spinach chloroplast genome, PetG, PetL, and PetN, all retained their initiating formylmethionine, while the nuclear encoded PetM was cleaved after import from the cytoplasm. While the sequences of PetG and PetN revealed no discrepancy with translations of the spinach chloroplast genome, Phe was detected at position 2 of PetL. The spinach chloroplast genome reports a codon for Ser at position 2 implying the presence of a DNA sequencing error or a previously undiscovered RNA editing event. Clearly, complete annotation of genomic data requires detailed expression measurements of primary structure by mass spectrometry. Full subunit coverage of an oligomeric intrinsic membrane protein complex by LCMS+ presents a new facet to intact mass proteomics.

Amino Acid Sequence↗