Biomedical subjects

W A Cramer

Publications and source records attributed to W A Cramer.

At least 19 recordsLinked to original sources

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↗

PubMed1995

On the physical basis for the cis-positive rule describing protein orientation in biological membranes.

The topology of hydrophobic intramembrane proteins is characterized by a statistical asymmetry in the distribution of positively-charged residues on the two sides of the membrane, the 'inside- or cis-positive rule'. A mechanism is proposed involving only neutral residue transfer. For a tightly bound polypeptide adsorbed on the membrane and not at equilibrium, the pK values of the ionic residues related to dissociation of the proton into the aqueous phase bulk are increased because of interaction with the negative charges at the membrane surface. The pK shift would selectively neutralize aspartate and glutamate residues, favoring their translocation across the membrane, while stabilizing the impermeant positively charged state of lysine and arginine residues.

Cell Membrane↗

PubMed1995

Structure-function of the channel-forming colicins.

The channel-forming colicins are plasmid-encoded bacteriocins that kill E. coli and related cells and whose mode of action is of interest in related problems of protein import and toxicology. Colicins parasitize metabolite receptors in the outer membrane and translocate across the periplasm with the aid of the Tol or Ton protein systems. X-ray structure data for the channel domain and colicin are available. Residues have been identified that affect the channel ion selectivity and particular helices implicated in channel structure and in conformational changes required for binding or insertion of the channel into the membrane. Unique aspects of the colicin channel system are the involvement of protein import in the gating process, the existence of multiple open and closed states, and the existence and action of an immunity protein that involves specific intramembrane helix-helix interactions with transmembrane helices of the colicin channel-forming domains.

Amino Acid Sequence↗

PubMed1994

Topography of the heme prosthetic group of cytochrome b-559 in the photosystem II reaction center.

The topography of the heme prosthetic group of cytochrome b-559 of the photosystem II reaction center was determined from measurement of the orientation of its alpha- and beta-polypeptides in thylakoid membranes of spinach chloroplasts and in osmotically disrupted cells of the cyanobacterium Synechocystis sp. PCC 6803. The accessibility to trypsin proteolysis of an epitope located near the solvent-exposed N-terminus of the beta-subunit was compared to that of the alpha-subunit, whose N- and C-termini had previously been localized from the trypsinolysis pattern to the stromal and lumenal sides of spinach thylakoid membranes, respectively [Tae et al. (1988) Biochemistry 27, 9075-9080; Vallon et al. (1989) Biochim. Biophys. Acta 975, 132-141]. The N-terminal epitope of the cyanobacterial beta-subunit was modified by introducing a tridecapeptide epitope, previously found to be immunoreactive, from the C-terminal region of the spinach chloroplast alpha-subunit. This epitope had no homology with the cyanobacterial alpha-subunit. The cells with the hybrid beta-subunit retained full photosynthetic activity. The intactness of membranes from osmotically shocked cyanobacteria was tested by trypsin inaccessibility to (a) the alpha-subunit C-terminus and (b) the manganese-stabilizing protein (MSP) of the oxygen-evolving complex that is on the lumenal side of the membrane. The loss after trypsinolysis of most of the beta-subunit immunoreactivity, under conditions where (i) the alpha-subunit was cleaved near the N-terminus in both spinach thylakoids and osmotically shocked cyanobacterial membranes and (ii) the MSP protein in cyanobacteria was not disrupted, implied that the orientation of the beta-subunit was parallel to that of the alpha-subunit in both kinds of membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Comparison of cytochrome b-559 content in photosystem II complexes from spinach and Synechocystis species PCC 6803.

Cytochrome b-559 is an integral component of photosystem II complexes from both plants and cyanobacteria. However, the number of cytochrome b-559 associated with the photosystem II reaction center has been the subject of controversy. Some studies have concluded that there is one heme equivalent of cytochrome b-559 per reaction center, some studies have found two, and some studies have reported intermediate values. Most of the previous experiments have used only one method to quantitate the antenna size of the preparation. In this study, we compare the cytochrome b-559 content in a cyanobacterial and a plant photosystem II preparation. The plant preparation is derived from spinach, and previous work has shown that it has an antenna size of approximately 100 chlorophylls [MacDonald, G. M., & Barry, B. A. (1992) Biochemistry 31, 9848-9856]. The cyanobacterial preparation is from Synechocystis sp. PCC 6803, and previous work has shown that it has an antenna size of approximately 60 chlorophylls [Noren, G. H., Boerner, R. J., & Barry, B. A. (1991) Biochemistry 30, 3943-3950]. Both preparations are isolated through the use of ion-exchange chromatography, and both preparations are monodisperse in the same nonionic detergent. In our comparative study, we quantitate antenna size by three different methods. Our work shows that, depending on the method used to estimate antenna size, the oxygen-evolving spinach photosystem II preparation contains 0.82-1.0 cytochrome b-559 per reaction center, while the oxygen-evolving cyanobacterial preparation contains 1.5-2.1 cytochrome b-559 per reaction center.

Cyanobacteria↗

PubMed1994

Characterization of the chloroplast cytochrome b6f complex as a structural and functional dimer.

Size analysis of the cytochrome b6f complex by FPLC Superose-12 chromatography and Blue Native PAGE indicated a predominantly dimeric component with M(r) = (1.9-2.5) x 10(5). The true dimer molecular weight including bound lipid, but not detergent, was estimated to be 2.3 x 10(5). Size and shape analysis by negative-stain single-particle electron microscopy indicated that the preparation of dimeric complexes contains a major population that has a protein cross section 40% larger than the monomer, binds more negative stain, and has a geometry with a distinct 2-fold axis of symmetry compared to the monomeric complex. The dimeric species is more stable at higher ionic strength with respect to conversion to the monomeric species. SDS-PAGE of monomer and dimer preparations indicated that both contain the four major polypeptides in approximately equal stoichiometry and also contain the petG M(r) 4000 subunit. One bound chlorophyll a per monomer, part of the bound lipid, is present in monomer and dimer. The in vitro electron-transport activity (decyl-PQH2-->PC-ferricyanide) of the separated dimer was comparable to that of the isolated b6f complex and was 4-5-fold greater than that of the monomer preparation, whose activity could be attributed to residual dimer. No difference in the properties of the dimer and monomer was detected by SDS-PAGE or redox difference spectrophotometry that could account for the difference in activities. However, the concentration of the Rieske [2Fe-2S] center was found by EPR analysis of the gy = 1.90 signal to be lower in the monomer fraction by a factor of 3.5 relative to the dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopolymers↗

PubMed1994

Crystal structure of chloroplast cytochrome f reveals a novel cytochrome fold and unexpected heme ligation.

BACKGROUND: Cytochrome f is the high potential electron acceptor of the chloroplast cytochrome b6f complex, and is the electron donor to plastocyanin. The 285-residue cytochrome f subunit is anchored in the thylakoid membrane of the chloroplast by a single membrane-spanning segment near the carboxyl terminus. A soluble redox-active 252-residue lumen-side polypeptide with native spectroscopic and redox properties, missing the membrane anchor and carboxyl terminus, was purified from turnip chloroplasts for structural studies. RESULTS: The crystal structure of cytochrome f, determined to 2.3 A resolution, has several unexpected features. The 252-residue polypeptide is organized into one large and one small domain. The larger heme-binding domain is strikingly different from known structures of other c-type cytochromes and has the same fold as the type III domain of the animal protein, fibronectin. Cytochrome f binds heme with an unprecedented axial heme iron ligand: the amino terminus of the polypeptide. CONCLUSION: The first atomic structure of a subunit of either the cytochrome b6f complex or of the related cytochrome bc1 complex has been obtained. The structure of cytochrome f allows prediction of the approximate docking site of plastocyanin and should allow systematic studies of the mechanism of intra- and inter-protein electron transfer between the cytochrome heme and plastocyanin copper, which are approximately isopotential. The unprecedented axial heme iron ligand also provides information on the sequence of events (i.e. cleavage of signal peptide and ligation of heme) associated with translocation of the cytochrome across the membrane and its subsequent folding.

Amino Acid Sequence↗

PubMed1994

On the nature of the unfolded intermediate in the in vitro transition of the colicin E1 channel domain from the aqueous to the membrane phase.

The transition of the colicin E1 channel polypeptide from a water-soluble to membrane-bound state occurs in vitro at acid pH values that are associated with an unfolded channel structure whose properties qualitatively resemble those of a "molten globule," or "compact unfolded," intermediate state. The role of such a state for activity was tested by comparing the pH dependence of channel-induced solute efflux and the amplitude of the near-UV CD spectrum. The requirement of a partly unfolded state for activity was shown by the coincidence of the onset of channel activity measured for 4 different lipid compositions with the decrease in near-UV CD amplitude as a function of pH. Tertiary constraints on the 3 tryptophans of the colicin channel, assayed by the amplitude of the near-UV CD spectrum, are retained over the pH range 3-4 where channel activity could be measured and, as well, at pH 2. In addition, the tryptophan fluorescence emission spectrum is virtually unchanged over the pH range 2-6. The temperature independence of the near-UV spectrum at pH 3-6 up to 70 degrees C implies that the colicin E1 channel polypeptide is more stable than that of colicin A. A transition between 53 and 58 degrees C in the amplitude of the near-UV CD is consistent with preservation of part of the hydrophobic core in a destabilized state at pH 2. Thus, the unfolded state associated with colicin activity at acidic pH has the properties of a "compact unfolded" state, having some, but not all of the properties of a "molten globule."(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗

PubMed1994

Crystallization and characterization of colicin E1 channel-forming polypeptides.

Crystals of the channel-forming domain of colicin E1 from E. coli were grown by vapor diffusion at pH 6.4 and higher pH values. Cleavage of the colicin molecule with trypsin or thermolysin produced two of the pore-forming polypeptides used in these experiments. The third polypeptide was purified from a constructed plasmid that overexpresses only the C-terminal domain of colicin E1. Polypeptide crystals are tetragonal with space group I4, have one monomer in the asymmetric unit, and diffract to 2.2-2.4 A. Unit cell parameters for the tryptic and thermolytic polypeptides are a = 102.9 A and c = 35.6 A. Crystals of the overexpressed polypeptide have unit cell parameters of a = 87.2 A and c = 59.1 A. The crystals were characterized by precession photography, and native data sets of each channel-forming fragment were collected on a Siemens-Nicolet area detector. The crystallization and characterization of these polypeptides are the first steps in the structure determination of the channel-forming domain of colicin E1.

Amino Acid Sequence↗

PubMed1994

Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.

Blue native Electrophoresis is a "charge shift" method developed for isolation of native membrane protein complexes from biological membranes that also separates both acidic and basic water-soluble proteins at a fixed pH of 7.5. In combination with a second dimension sodium dodecylsulfate electrophoresis it provides an analytical method for the determination of molecular mass and oligomeric state of nondissociated complexes, of subunit composition, and of degree of purity and for the detection of subcomplexes. The method was applied to analysis of cytochrome bc/bf complexes. By combination of a novel colorless native polyacrylamide gel electrophoresis (CN-PAGE) with blue native BN-PAGE, a two-dimensional native technique was developed that is suitable for preparation of highly pure membrane protein complexes.

Animals↗

PubMed1994

Structural aspects of the cytochrome b6f complex; structure of the lumen-side domain of cytochrome f.

The following findings concerning the structure of the cytochrome b6f complex and its component polypeptides, cyt b6, subunit IV and cytochrome f subunit are discussed: (1) Comparison of the amino acid sequences of 13 and 16 cytochrome b6 and subunit IV polypeptides, respectively, led to (a) reconsideration of the helix lengths and probable interface regions, (b) identification of two likely surface-seeking helices in cyt b6 and one in SU IV, and (c) documentation of a high degree of sequence invariance compared to the mitochondrial cytochrome. The extent of identity is particularly high (88% for conserved and pseudoconserved residues) in the segments of cyt b6 predicted to be extrinsic on the n-side of the membrane. (2) The intramembrane attractive forces between trans-membrane helices that normally stabilize the packing of integral membrane proteins are relatively weak. (3) The complex isolated in dimeric form has been visualized, along with isolated monomer, by electron microscopy. The isolated dimer is much more active than the monomer, is the major form of the complex isolated and purified from chloroplasts, and is inferred to be a functional form in the membrane. (4) The isolated cyt b6f complex contains one molecule of chlorophyll a. (5) The structure of the 252 residue lumen-side domain of cytochrome f isolated from turnip chloroplasts has been solved by X-ray diffraction analysis to a resolution of 2.3 A.

Amino Acid Sequence↗

PubMed1993

Intramembrane helix-helix interactions as the basis of inhibition of the colicin E1 ion channel by its immunity protein.

It had previously been hypothesized that the ability of a small number of immunity protein molecules in the cytoplasmic membrane to confer protection against the lethal effects of a channel-forming colicin involves a complex stabilized by electrostatic or polar interactions between immunity protein, the colicin channel, and specific sites on the cytoplasmic membrane surface defined by the presence of the tol gene translocation proteins. The hypothesis was tested (a) by constructing a hybrid colicin molecule, IaE1, containing the E1 channel domain, and the translocation and receptor domains of Ia, and (b) by altering charged residues in all peripheral regions of the immunity protein to neutral residues. It was concluded that the specificity of immunity protein requires neither specific translocation proteins, nor a specific arrangement of charged residues in the immunity protein. (c) In addition, by making 65 site-directed mutations, "immunity by-pass" mutants were found at five different loci, Ala474, Ser477, His440, Phe443, and Gly444, on two proposed membrane-spanning helices of the open colicin channel, one hydrophobic (A471-A488) and one amphiphilic (V441-W460). The mutants in the hydrophobic helix showed a larger bypass effect. The "bypass" phenotype could be assayed by (i) cytotoxicity and (ii) K+ efflux in imm+ cells caused by a bypass mutant but not wild-type colicin. It is concluded that the immunity protein exerts its specific effect through rapid lateral diffusion in the cytoplasmic membrane and helix-helix recognition and interaction with at least one hydrophobic and one amphiphilic trans-membrane helix of the colicin channel. Interaction with the amphiphilic helix implies that the immunity protein can react with the channel in the open state.

Amino Acid Sequence↗

PubMed1993

Overproduction and purification of the colicin E1 immunity protein.

Colicin E1 immunity protein encoded by the plasmid colicin E1 was overproduced in Escherichia coli from an expression plasmid which was constructed by placing the immunity protein-encoding sequence downstream of the tac promoter and by properly positioning the ribosome binding site on a run-away replication vector. The immunity protein was solubilized with 0.5% Brij 58 from the membrane fraction and purified to homogeneity by a simple batch procedure with hydroxyapatite gel and reverse-phase chromatography. A 15-residue NH2-terminal amino acid sequence was determined to be the same as that deduced from the DNA sequence. The effect of the purified immunity protein on membranes was tested in vitro using solute-loaded liposomes. The immunity protein added to the liposomes showed a small but significant channel or lytic activity that is an indicator of its hydrophobic nature.

Base Sequence↗

PubMed1993

The redox properties of cytochromes b imposed by the membrane electrostatic environment.

The effect of the dipole potential field of extended membrane spanning alpha-helices on the redox potentials of b cytochromes in energy transducing membranes has been calculated in the context of a three phase model for the membrane. In this model, the membrane contains three dielectric layers; (i) a 40-A hydrophobic membrane bilayer, with dielectric constant em = 3-4, (ii) 10-20-A interfacial layers of intermediate polarity, ein = 12-20, that consist of lipid polar head groups and peripheral protein segments, and (iii) an external infinite water medium, ew = 80. The unusually positive midpoint potential, Em = +0.4 V, of the "high potential" cytochrome b-559 of oxygenic photosynthetic membranes, a previously enigmatic property of this cytochrome, can be explained by (i) the position of the heme in the positive dipole potential region near the NH2 termini of the two parallel helices that provide its histidine ligands, and (ii) the loss of solvation energy of the heme ion due to the low dielectric constant of its surroundings, leading to an estimate of +0.31 to +0.37 V for the cytochrome Em. The known tendency of this cytochrome to undergo a large -delta Em shift upon exposure of thylakoid membranes to proteases or damaging treatments is explained by disruption of the intermediate polarity (ein) surface dielectric layer and the resulting contact of the heme with the external water medium. Application of this model to the two hemes (bn and bp) of cytochrome b of the cytochrome bc1 complex, with the two hemes placed symmetrically in the low dielectric (em) membrane bilayer, results in Em values of hemes bn and bp that are, respectively, somewhat too negative (approximately -0.1 V), and much too positive (approximately +0.3 V), leading to a potential difference, Em(bp) - Em(bn), with the wrong sign and magnitude, +0.25 V instead of -0.10 to -0.15 V. The heme potentials can only be approximately reconciled with experiment, if it is assumed that the two hemes are in different dielectric environments, with that of heme bp being more polar.

Amino Acid Sequence↗

PubMed1992

Truncation of the COOH-terminal domain of the psbE gene product in Synechocystis sp. PCC 6803: requirements for photosystem II assembly and function.

The COOH-terminal domain of the 80-residue cytochrome b559 alpha-subunit (psbE gene product) in Synechocystis sp. PCC 6803 was sequentially truncated in order to determine the minimum polypeptide length needed for function and assembly. A stop codon was introduced into the Arg-50, Arg-59, or Tyr-69 codons of the psbE gene, generating mutants truncated by 31, 22, and 12 residues, respectively. Removal of 12 residues caused a decrease of 20% in PSII function. Truncation of 22 or 31 residues caused a large decrease (60-85%) in the photoautotrophic growth rate, the rate of O2 evolution, and the amplitude of the 77 K 696-nm fluorescence, and a concomitant increase in the constant yield fraction (F0/Fmax) of the chlorophyll fluorescence. The level of residual activity in the Arg50-stop mutant was 10-20% of the wild type, which was reflected in a similar low level of immunochemically detected D2 polypeptide. Quantitation of the PSII reaction center stoichiometry of the Arg50-stop mutant by analysis of [14C]DCMU binding also showed a 5-fold decrease (1:910 Chl in wild type and 1:5480 Chl in R50) in the PSII reaction center concentration. However, the KD value for DCMU in the residual 15% of the complexes to which it bound was approximately equal to that (25 nM) of the wild type. Northern blot analysis showed no decrease in the b559 psbE mRNA level. Chemical difference spectral analysis of heme content indicated that the level of native cytochrome b559 heme in the Arg50-stop mutant (1:640 Chl) was 80% that of wild type (1:510 Chl).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Constraints imposed by protease accessibility on the trans-membrane and surface topography of the colicin E1 ion channel.

The surface topography of a 190-residue COOH-terminal colicin E1 channel peptide (NH2-Met 333-Ile 522-COOH) bound to uniformly sized 0.2-micron liposomes was probed by accessibility of the peptide to proteases in order (1) to determine whether the channel structure contains trans-membrane segments in addition to the four alpha-helices previously identified and (2) to discriminate between different topographical possibilities for the surface-bound state. An unfolded surface-bound state is indicated by increased trypsin susceptibility of the bound peptide relative to that of the peptide in aqueous solution. The peptide is bound tightly to the membrane surface with Kd < 10(-7) M. The NH2-terminal 50 residues of the membrane-bound peptide are unbound or loosely bound as indicated by their accessibility to proteases, in contrast with the COOH-terminal 140 residues, which are almost protease inaccessible. The general protease accessibility of the NH2-terminal segment Ala 336-Lys 382 excludes any model for the closed channel state that would include trans-membrane helices on the NH2-terminal side of Lys 382. Lys 381-Lys 382 is a major site for protease cleavage of the surface-bound channel peptide. A site for proteinase K cleavage just upstream of the amphiphilic gating hairpin (K420-K461) implies the presence of a surface-exposed segment in this region. These protease accessibility data indicate that it is unlikely that there are any alpha-helices on the NH2-terminal side of the gating hairpin K420-K461 that are inserted into the membrane in the absence of a membrane potential. A model for the topography of an unfolded monomeric surface-bound intermediate of the colicin channel domain, including a trans-membrane hydrophobic helical hairpin and two or three long surface-bound helices, is proposed.

Amino Acid Sequence↗