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W A Cramer

Publications and source records attributed to W A Cramer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Comparison of the cytochrome bc1 complex with the anticipated structure of the cytochrome b6f complex: Le plus ça change le plus c'est la même chose.

Structural alignment of the integral cytochrome b6-SU IV subunits with the solved structure of the mitochondrial bc1 complex shows a pronounced asymmetry. There is a much higher homology on the p-side of the membrane, suggesting a similarity in the mechanisms of intramembrane and interfacial electron and proton transfer on the p-side, but not necessarily on the n-side. Structural differences between the bc1 and b6f complexes appear to be larger the farther the domain or subunit is removed from the membrane core, with extreme differences between cytochromes c1 and f. A special role for the dimer may involve electron sharing between the two hemes b(p), which is indicated as a probable event by calculations of relative rate constants for intramonomer heme b(p) --> heme b(n), or intermonomer heme b(p) --> heme b(p) electron transfer. The long-standing observation of flash-induced oxidation of only approximately 0.5 of the chemical content of cyt f may be partly a consequence of the statistical population of ISP bound to cytfon the dimer. It is proposed that the p-side domain of cyt f is positioned with its long axis parallel to the membrane surface in order to: (i) allow its large and small domains to carry out the functions of cyt c1 and suVIII, respectively, of the bc1 complex, and (ii) provide maximum dielectric continuity with the membrane. (iii) This position would also allow the internal water chain ("proton wire") of cyt f to serve as the p-side exit port for an intramembrane H+ transfer chain that would deprotonate the semiquinol located in the myxothiazol/MOA-stilbene pocket near heme b(p). A hypothesis is presented for the identity of the amino acid residues in this chain.

Animals↗
PubMed1998

Perturbation of the internal water chain in cytochrome f of oxygenic photosynthesis: loss of the concerted reduction of cytochromes f and b6.

The 1.96 A structure of turnip cytochrome f revealed a linear internal chain of H2O molecules with the oxygen atoms of the chain having occupancies and "B" factors comparable to those of neighboring atoms [Martinez et al. (1996) Protein Sci. 5, 1081-1092. ]. Four waters extend 11 A from the heme toward Lys66 on the cytochrome surface. All residues that contribute an atom to the 15 H-bonds of five internal H2O molecules are essentially conserved in 23 cytochrome sequences. With only Gln and Asn side chains involved in H-bonding, the water chain resembles a "proton wire". The function of the conserved H2O chain was tested through site-directed mutagenesis of these Asn and Gln residues. Four of the five conserved Asn/Gln residues were changed in six mutants generated in the green alga, Chlamydomonas reinhardtii. Except for the N168F mutant, all grew photosynthetically. Although the rates of oxidation of cyt f oxidation and of reduction of cyt b6 (5-6 ms in the wild type) were not significantly affected, the rates of cyt f reduction and generation of the slow electrochromic band shift (Deltapsis) were markedly decreased, the half-times increasing to as much as 38 and 18 ms, respectively. Thus, in these mutants, reduction of cyt b6 reduction clearly precedes that of cyt f. Retardation of Deltapsis in the absence of an observable change in the rate of cyt b6 reduction implied that the rate of H+ translocation decreased in the mutants, and electron transfer was concomitantly retarded, most likely between the ISP and cyt f. The following was concluded: (i) proton and electron transfer are coupled in reduction of cyt f, and the cyt f water chain functions in H+ transfer; (ii) reduction of the high- and low-potential chains in the b6f complex is not concerted in the water chain mutants; and (iii) quinol deprotonation and electron transfer from reduced quinone are initiated by an early event, probably the movement of the ISP triggered by oxidation of cyt f.

Animals↗
PubMed1998

Purification and characterization of monomeric Escherichia coli vitamin B12 receptor with high affinity for colicin E3.

The btuB gene product from Escherichia coli is a 66.5-kDa integral outer membrane protein required for high-affinity uptake of cyanocobalamin and the translocation of E group colicins and colicin A. Efficient purification of overexpressed BtuB containing stoichiometric levels of bound lipopolysaccharide has been achieved through the extraction of the outer membrane with nonionic detergent followed by ion-exchange chromatography. Analysis of far UV circular dichroism spectra indicates a predominantly beta-sheet secondary structure (76 +/- 4%) with a low alpha-helical content (15 +/- 3%), providing the first direct evidence for secondary structure models derived from sequence and hydropathy analysis. Characterization of the octylglucoside-solubilized receptor by sedimentation equilibrium and sedimentation velocity analysis reveals a monodisperse protein-detergent complex of approximately 89 kDa with a sedimentation coefficient of 4.7 S which, after correction for bound detergent, indicates that BtuB is purified as a monomer. BtuB binds vitamin B12 with a stoichiometry of approximately 1:1, as observed by a shift in the sedimentation profile of the vitamin to the much faster velocity observed for the protein-detergent complex. The preincubation of colicin E3 with stoichiometric levels of BtuB protects susceptible strains from the lethal effects of the colicin and results in a complex with a sedimentation coefficient appropriate for a BtuB-detergent-colicin E3 complex, demonstrating that monomeric BtuB retains high affinity for this particular ligand after isolation.

Bacterial Outer Membrane Proteins↗
PubMed1998

Identification of the basic residues of cytochrome f responsible for electrostatic docking interactions with plastocyanin in vitro: relevance to the electron transfer reaction in vivo.

The prominent basic patch seen in the atomic structure of the lumen-side domain of turnip cytochrome f, consisting of Arg209 and Lys187, 58, 65, and 66, was proposed to be an electrostatically complementary docking site for its physiological electron acceptor, plastocyanin [Martinez, S. E., Huang, D., Szczepaniak, A., Cramer, W. A., and Smith, J. L. (1994) Structure 2, 95-105]. This proposal agrees with solution studies on the cytochrome f/plastocyanin electron-transfer reaction that showed a major contribution of electrostatic interactions to the docking, but not with studies on the oxidation rate of cyt f in vivo using mutants in which the basic patch of cyt f was neutralized. The apparent contradiction might be explained by an unknown electron acceptor protein for cyt f. However, (i) flash-induced oxidation of cyt f is absent in a PC-deficient mutant. (ii) Lys58, 65, and 66 in the large domain and Lys188 and 189 in the small domain are major contributors to the ionic strength dependence of the electron-transfer reaction in solution. Replacement of Lys58 and 65 by neutral residues and of Lys66 by the acidic residue Glu66 resulted in a >10-fold decrease in the rate of electron transfer in solution and complete loss of its ionic strength dependence. Replacement of Lys188 and Lys189 in the small domain of cyt f resulted in a 3-4-fold decrease in the second-order rate constant and a smaller dependence of the overall rate of electron transfer on ionic strength, corresponding to a loss of two positive charges. (iii) Acidification of the thylakoid lumen cannot explain the absence of electrostatic interactions. (iv) Changing the five lysines to acidic residues did not result in any significant retardation of the rate of cyt f oxidation in vivo. If the docking of cyt f and plastocyanin in vivo is mediated by basic residues of cyt f, they are different from those that mediate electron transfer in vitro or that are implicated by simulations of electrostatic interactions of the docking. Alternatively, docking of cyt f/PC in vivo is limited by spatial constraints or release of PC from P700 that precludes a rate-limiting mediation of the cyt f/PC reaction by specific electrostatic interactions. The cyt f/PC system in Chlamydomonas reinhardtii is the first electron-transfer couple for which the role of electrostatics in mediating the docking reaction has been studied both in vitro and in vivo.

Animals↗
PubMed1998

Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array.

Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the alpha-helical content increases from 60-64% to 80-90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3- to 4-fold increase in the average area subtended per molecule to 4,200 A2 on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled alpha-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.

Calorimetry, Differential Scanning↗
PubMed1998

Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers.

The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.

Amino Acid Sequence↗
PubMed1998

Coupled plasmon-waveguide resonance spectroscopy studies of the cytochrome b6f/plastocyanin system in supported lipid bilayer membranes.

The incorporation of cytochrome (cyt) b6f into a solid-supported planar egg phosphatidylcholine (PC) bilayer membrane and complex formation with plastocyanin have been studied by a variant of surface plasmon resonance called coupled plasmon-waveguide resonance (CPWR) spectroscopy, developed in our laboratory. CPWR combines greatly enhanced sensitivity and spectral resolution with direct measurement of anisotropies in refractive index and optical extinction coefficient, and can therefore probe structural properties of lipid-protein and protein-protein interactions. Cyt b6f incorporation into the membrane proceeds in two stages. The first occurs at low protein concentration and is characterized by an increase in total proteolipid mass without significant changes in the molecular order of the system, as demonstrated by shifts of the resonance position to larger incident angles without changing the refractive index anisotropy. The second stage, occurring at higher protein concentrations, results in a decrease in both the mass density and the molecular order of the system, evidenced by shifts of the resonance position to smaller incident angles and a large decrease in the membrane refractive index anisotropy. Plastocyanin can bind to such a proteolipid system in three different ways. First, the addition of plastocyanin before the second stage of b6f incorporation begins results in complex formation between the two proteins with a KD of approximately 10 microM and induces structural changes in the membrane that are similar to those occurring during the second stage of complex incorporation. The addition of larger amounts of plastocyanin under these conditions leads to nonspecific binding to the lipid phase with a KD of approximately 180 microM. Finally, the addition of plastocyanin after the completion of the second phase of b6f incorporation results in tighter binding between the two proteins (KD approximately 1 microM). Quantitation of the binding stoichiometry indicates that two plastocyanin molecules bind tightly to the dimeric form of the cyt b6f complex, assuming random insertion of the cytochrome into the bilayer. The structural basis for these results and formation of the proteolipid membrane are discussed.

Cyanobacteria↗
PubMed1997

Biological identity and diversity in photosynthesis and respiration: structure of the lumen-side domain of the chloroplast Rieske protein.

BACKGROUND: The cytochrome b6f complex functions in oxygenic photosynthesis as an integral membrane protein complex that mediates coupled electron transfer and proton translocation. The Rieske [2Fe-2S] protein subunit of the complex functions at the electropositive (p) membrane interface as the electron acceptor for plastoquinol and donor for the cytochrome f subunit, and may have a dynamic role in catalyzing electron and proton transfer at the membrane interface. There are significant structure/function similarities to the cytochrome bc1 complex of the respiratory chain. RESULTS: The 1.83 A crystal structure of a 139-residue C-terminal fragment of the Rieske [2Fe-2S] protein, derived from the cytochrome b6f complex of spinach chloroplasts, has been solved by multiwavelength anomalous diffraction. The structure of the fragment comprises two domains: a small 'cluster-binding' subdomain and a large subdomain. The [2Fe-2S] cluster-binding subdomains of the chloroplast and mitochondrial Rieske proteins are virtually identical, whereas the large subdomains are strikingly different despite a common folding topology. A structure-based sequence alignment of the b6f and bc1 groups of Rieske soluble domains is presented. CONCLUSIONS: The segregation of structural conservation and divergence in the cluster-binding and large subdomains of the Rieske protein correlates with the overall relatedness of the cytochrome b6f and bc1 complexes, in which redox domains in the aqueous p phase are dissimilar and those within the membrane are similar. Distinct sequences and surface charge distributions among Rieske large subdomains may provide a signature for interaction with the p-side oxidant protein and for the pH of the intraorganelle compartment.

Amino Acid Sequence↗
PubMed1997

Critical analysis of the extinction coefficient of chloroplast cytochrome f.

In oxygenic photosynthesis the cytochrome bf complex links electron transport between photosystem II and photosystem I. The largest subunit of the complex is cytochrome f, a 32-kDa polypeptide that is anchored in the membrane by a transmembrane alpha helix located near the carboxyl end. The three-dimensional structure of the soluble domain of cytochrome f isolated from turnip has been determined by X-ray crystallography to 1.96 A resolution. The structure revealed several novel features compared to previously solved soluble c-type cytochrome structures, including a predominant beta-strand motif, the N-terminal alpha-amino group of a tyrosyl residue as an orthogonal ligand, and a bound internal water chain. Here we report a novel and unprecedented extinction coefficient for cytochrome f. Using the pyridine hemochrome assay, the reduced minus oxidized extinction coefficient for the soluble domain of turnip cytochrome f was 26 +/- 1 mM-1 cm-1 for the alpha-band wavelength peak at 554 nm relative to the isosbestic wavelengths (534, 543.5 and 560.5 nm), and 25 +/- 1 mM-1 cm-1 for spinach cytochrome f relative to the isosbestic wavelengths (533.5, 543.3 and 560.2). The extinction coefficients reported here are significantly higher than previously published values for cytochrome f. We believe earlier determinations underestimated the cytochrome f extinction coefficient and that the same is likely true for commonly used extinction coefficients of cytochrome b6. The cytochrome f extinction coefficient is large compared to most other c-type cytochromes, which could be due to the unique axial ligand of the cytochrome f heme. Polarographic measurements show the midpoint potential of soluble turnip cytochrome f to be 362 +/- 5 mV at pH 7.5. The midpoint potential was pH-independent from 5.0 to 8.5, and pH-dependent from pH 8.5 to 10.5 (-58 mV/pH unit) with a pK on the oxidized from near 9. Storage of some samples of purified turnip and spinach cytochrome f at -20 degrees C modified the heme environment in a fraction of the protein, shifting the midpoint potential to near -165 mV (pH 7.5) and the peak of the alpha-band absorption spectrum from 554 nm to 552 nm.

Chloroplasts↗
PubMed1997

A mechanism for toxin insertion into membranes is suggested by the crystal structure of the channel-forming domain of colicin E1.

BACKGROUND: Channel-forming colicins, including colicin E1, are a sub-family of bacteriocins. The toxic action of colicin E1 is derived from its ability to form a voltage-gated channel, which causes depolarization of the cytoplasmic membrane of sensitive Escherichia coli cells. In this process, the toxin-like colicin E1 molecule must undergo a substantial structural transition from a soluble state, in which it binds the target cell, to a membrane-bound state. Details of the structural changes that accompany this conversion may be directly applicable to other channel-forming toxins, as well as to the mechanism by which proteins insert into or cross membranes. RESULTS: The structure of the 190-residue channel-forming domain of colicin E1 in its soluble form has been solved at 2.5 A resolution. This structure contains 10alpha helices arranged in three layers (A-C) with a central hydrophobic helical hairpin in layer B, which is proposed to anchor the membrane-bound form in the bilayer. The extended N-terminal helix I provides a connection to the rest of the colicin E1 molecule, and the loop I-II may act as a hinge for re-orientation of the domain for membrane binding. A set of conserved positively charged residues on layer C may provide the docking surface on the molecule for membrane attachment. A large internal cavity between layers B and C may allow these layers to disengage, suggesting a mechanism for unfolding the molecule on the membrane that involves the perturbation of the interhelical hydrophobic interactions in layer C. CONCLUSION: On the basis of the structure of the colicin E1 channel-forming domain, its comparison with the structure of the colicin A domain and the known requirement for initial electrostatic and subsequent hydrophobic interactions, molecular details of the docking, unfolding and insertion of the channel-forming domain into the membrane are proposed. The model for docking and initial interaction with the membrane positions the hydrophobic hairpin 'anchor' approximately parallel to the membrane surface. Hydrophobic interactions in the docking layer may then be displaced by interactions with the membrane, spreading the helices on the surface and exposing the hydrophobic hairpin for insertion into the membrane.

Amino Acid Sequence↗
PubMed1997

Electrostatic effects on electron-transfer kinetics in the cytochrome f-plastocyanin complex.

In a complex of two electron-transfer proteins, their redox potentials can be shifted due to changes in the dielectric surroundings and the electrostatic potentials at each center caused by the charged residues of the partner. These effects are dependent on the geometry of the complex. Three different docking configurations (DCs) for intracomplex electron transfer between cytochrome f and plastocyanin were studied, defined by 1) close contact of the positively charged region of cytochrome f and the negatively charged regions of plastocyanin (DC1) and by (2, 3) close contact of the surface regions adjacent to the Fe and Cu redox centers (DC2 and DC3). The equilibrium energetics for electron transfer in DC1-DC3 are the same within approximately +/-0.1 kT. The lower reorganization energy for DC2 results in a slightly lower activation energy for this complex compared with DC1 and DC3. The long heme-copper distance (approximately 24 A) in the DC1 complex drastically decreases electronic coupling and makes this complex much less favorable for electron transfer than DC2 or DC3. DC1-like complexes can only serve as docking intermediates in the pathway toward formation of an electron-transfer-competent complex. Elimination of the four positive charges arising from the lysine residues in the positive patch of cytochrome f, as accomplished by mutagenesis, exerts a negligible effect (approximately 3 mV) on the redox potential difference between cyt f and PC.

Biophysical Phenomena↗
PubMed1997

The TolA protein interacts with colicin E1 differently than with other group A colicins.

The 421-residue protein TolA is required for the translocation of group A colicins (colicins E1, E2, E3, A, K, and N) across the cell envelope of Escherichia coli. Mutations in TolA can render cells tolerant to these colicins and cause hypersensitivity to detergents and certain antibiotics, as well as a tendency to leak periplasmic proteins. TolA contains a long alpha-helical domain which connects a membrane anchor to the C-terminal domain, which is required for colicin sensitivity. The functional role of the alpha-helical domain was tested by deletion of residues 56 to 169 (TolA delta1), 166 to 287 (TolA delta2), or 54 to 287 (TolA delta3) of the alpha-helical domain of TolA, which removed the N-terminal half, the C-terminal half, or nearly the entire alpha-helical domain of TolA, respectively. TolA and TolA deletion mutants were expressed from a plasmid in an E. coli strain producing no chromosomally encoded TolA. Cellular sensitivity to the detergent deoxycholate was increased for each deletion mutant, implying that more than half of the TolA alpha-helical domain is necessary for cell envelope stability. Removal of either the N- or C-terminal half of the alpha-helical domain resulted in a slight (ca. 5-fold) decrease in cytotoxicity of the TolA-dependent colicins A, E1, E3, and N compared to cells producing wild-type TolA when these mutants were expressed alone or with TolQ, -R, and -B. In cells containing TolA delta3, the cytotoxicity of colicins A and E3 was decreased by a factor of >3,000, and K+ efflux induced by colicins A and N was not detectable. In contrast, for colicin E1 action on TolA delta3 cells, there was little decrease in the cytotoxic activity (<5-fold) or the rate of K+ efflux, which was similar to that from wild-type cells. It was concluded that the mechanism(s) by which cellular uptake of colicin E1 is mediated by the TolA protein differs from that for colicins A, E3, and N. Possible explanations for the distinct interaction and unique translocation mechanism of colicin E1 are discussed.

Amino Acid Sequence↗
PubMed1996

Characterization and crystallization of the lumen side domain of the chloroplast Rieske iron-sulfur protein.

A soluble, 139-residue COOH-terminal polypeptide fragment of the Rieske iron-sulfur protein of the cytochrome b6f complex from spinach chloroplasts was obtained by limited proteolysis of the complex and a two-step chromatography purification protocol. The purified Rieske iron-sulfur protein fragment was characterized by: (i) a single NH2-terminal sequence, NH2-Phe-Val-Pro-Pro-Gly-Gly, starting with residue 41 of the intact Rieske protein; (ii) a single molecular weight species determined by mass spectrometry with a molecular weight of 14,620 +/- 2 without the [2Fe-2S] cluster; (iii) an optical absorbance spectrum with redox- and pH-dependent maxima and minima; and (iv) a reduced-oxidized optical difference spectrum characterized by DeltaepsilonmM = 3.8 mM-1 cm-1 for DeltaA at 394 versus 409 nm, which was used to determine the midpoint oxidation-reduction potential, which is +359 +/- 7 mV at 25 degrees C from pH 5.5-6.5, and +319 +/- 2 mV at pH 7, with an apparent pKox = 6.5 +/- 0.2 for the oxidized protein. The EPR spectrum measured at 17 K was characterized by the g values, gz = 2.03 and gy = 1.90, and a broad band centered at gx approximately 1.74, very similar or identical to those of the Rieske cluster in the b6f complex, implying that the environment of the [2Fe-2S] cluster is similar to that in the complex. Midpoint potential determination by low temperature EPR yielded a redox midpoint potential (Em) of +365-375 mV of the soluble Rieske fragment at pH 6 and 7 and an Em of +295-300 mV of the Rieske cluster in the cytochrome b6f complex at pH 6 and 7. The Em difference implies that the environment of the cluster in the soluble Rieske fragment is slightly more polar than that of the cluster in the intact complex. Single crystals of the Rieske polypeptide were obtained that are capable of x-ray diffraction to atomic resolution (<2.5 A), contain one molecule per asymmetric unit, a solvent content of approximately 30%, and belong to the triclinic space group P1 with cell dimensions, a = 29.1 A, b = 31.9 A, c = 35.8 A, alpha = 95.6 degrees, beta = 107.1 degrees, gamma = 117.3 degrees.

Amino Acid Sequence↗
PubMed1996

Effect of the interdomain basic region of cytochrome f on its redox reactions in vivo.

The prominent interdomain basic surface region seen in the high-resolution structure of the active lumen-side C-terminal fragment of turnip cytochrome f, containing the conserved Lys58,65,66 (large domain) and Lys187 (small domain), has been inferred from in vitro studies to be responsible for docking of its physiological oxidant, plastocyanin. The effect of the putative docking region of cyt f on its reactivity in vivo was tested by site-directed mutagenesis in Chlamydomonas reinhardtii. Three charge-neutralizing mutants were constructed involving: (i)the two lysines (Lys188Asn-Lys189Gln) in the small domain, (ii) the three lysines (Lys58Gln-Lys65Ser-Lys66Glu) in the large domain, and (iii) all five of these lysines spanning both domains. All mutants grew phototrophically. The mutants displayed a 20-30% increase in average generation time, and comparable decreases in rates of steady-state oxygen evolution and the slow (millisecond) electrochromic 515 nm band shift. The magnitude of the changes was greatest in the 5-fold Lys-minus mutant (Lys58Gln-Lys65Ser-Lys66Glu-Lys188Asn-Lys189G ln). The mutants showed a small increase (approximately 25%) in the t1/2, from 0.2 to 0.25 ms, of cyt f photooxidation, far less than anticipated (ca. 100-fold) from in vitro studies of the effect of high ionic strength on the cyt f-PC interaction. The t1/2 of cyt f dark reduction via the Rieske protein increased from 5-6 ms in the wild type to 11-12 ms in the 5-fold Lys-minus mutant. Cells grown phototrophically in the absence of Cu, where cyt c6 is the electron acceptor of cyt f, displayed net rates of cytochrome photooxidation that were slightly faster than those in the presence of Cu, which also decreased by a factor of < or = 25% in the Lys-minus mutants. It was concluded that (a) the net effect of electrostatic interaction between cytochrome f and its electron acceptor in vivo is much smaller than measured in vitro and is not rate-limiting. This may be a consequence of a relatively high ionic strength environment and the small diffusional space available for collision and docking in the internal thylakoid lumen of log phase C. reinhardtii. (b) The efficiency of electron transfer to cytochrome f from the Rieske protein is slightly impaired by the neutralization of the lysine-rich domain.

Animals↗
PubMed1996

Characterization of electrostatic and nonelectrostatic components of protein--membrane binding interactions.

A general method was developed to determine the thermodynamic parameters for the interaction of protein with membranes. Protein intrinsic tryptophan fluorescence was quenched by titration with large unilamellar vesicles containing 9,10-dibrominated distearoylphosphatidylcholine (Br4-DSPC) or a small amount of trinitrophenylphosphatidylethanolamine (TNP-PE), Binding was modeled as a bimolecular reaction of free protein with a unit of "n" lipid molecules and a dissociation constant, Kd. The contribution of residual fluorescence and light scattering could be eliminated by using the second derivative of the titration function as the basis for calculations. For the binding of C-terminal channel domain polypeptides(178-190 residues) of the colicin El ion channel, n=50-60 and Kd=2-3 nM at pH 4, ionic strength, I=0.12 M, and anionic lipid content = 40% (surface potential, psi o =-30 mV), conditions for which the protein has high activity. Values of n = 95 and 210 for the binding of a C-terminal 293-residue colicin fragment and the 522 residue intact colicin E1 molecule scale qualitatively according to the increase in molecular size. General methods are presented to distinguish the electrostatic (delta G el) and nonelectrostatic (delta G nel) components of the total delta G for binding. Using Br4DSPC as the quencher, the binding of the channel polypeptide, P178, was characterized by delta G approximately -9.8 kcal/mol, and delta G el approximately -7.0 kcal/mol, and delta G el= -2.8 kcal/mol (psi o = -30 mV). Using TNP-PE as the quencher, similar values of delta G approximately -9.3 to -9.9 kcal/mol were determined, a somewhat smaller value for delta G nel approximately -5.0 kcal/mol, and a correspondingly larger value for deltaGnel approximately -4.9 kcal/mol. The existence of a delta G nel component of this magnitude may distinguish proteins that have the potential to insert into the membrane.

Colicins↗
PubMed1996

The heme redox center of chloroplast cytochrome f is linked to a buried five-water chain.

The crystal structure of the 252-residue lumen-side domain of reduced cytochrome f, a subunit of the proton-pumping integral cytochrome b6f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 A from crystals cooled to -35 degrees. The model was refined to an R-factor of 15.8% with a 0.013-A RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20 degrees, the structure at -35 degrees has a small change in relative orientation of the two folding domains and significantly lower isotropic temperature factors for protein atoms. The structure revealed an L-shaped array of five buried water molecules that extend in two directions from the N delta 1 of the heme ligand His 25. The longer branch extends 11 A within the large domain, toward Lys 66 in the prominent basic patch at the top of the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature factors are comparable to those of protein atoms. Virtually all residues that form hydrogen bonds with the water chain are invariant among 13 known cytochrome f sequences. The water chain has many features that optimize it as a proton wire, including insulation from the protein medium. It is suggested that this chain may function as the lumen-side exit port for proton translocation by the cytochrome b6f complex.

Chloroplasts↗
PubMed1996

Membrane binding of the colicin E1 channel: activity requires an electrostatic interaction of intermediate magnitude.

In vitro channel activity of the C-terminal colicin E1 channel polypeptide under conditions of variable electrostatic interaction with synthetic lipid membranes showed distinct maxima with respect to pH and membrane surface potential. The membrane binding energy was determined from fluorescence quenching of the intrinsic tryptophans of the channel polypeptide by liposomes containing N-trinitrophenyl-phosphatidylethanolamine. Maximum in vitro colicin channel activity correlated with an intermediate magnitude of the electrostatic interaction. For conditions associated with maximum activity (40% anionic lipid, I = 0.12 M, pH 4.0), the free energy of binding was delta G approximately -9 kcal/mol, with nonelectrostatic and electrostatic components, delta Gnel approximately -5 kcal/mol and delta Gel approximately -4 kcal/mol, and an effective binding charge of +7 at pH 4.0. Binding of the channel polypeptide to negative membranes at pH 8 is minimal, whereas initial binding at pH 4 followed by a shift to pH 8 causes only 3-10% reversal of binding, implying that it is kinetically trapped, probably by a hydrophobic interaction. It was inferred that membrane binding and insertion involves an initial electrostatic interaction responsible for concentration and binding to the membrane surface. This is followed by insertion into the bilayer driven by hydrophobic forces, which are countered in the case of excessive electrostatic binding.

Binding Sites↗