Binding of transducin and transducin-derived peptides to rhodopsin studies by attenuated total reflection-Fourier transform infrared difference spectroscopy

Fourier transform infrared difference spectroscopy combined with the attenuated total reflection technique allows the monitoring of the association of transducin with bovine photoreceptor membranes in the dark. Illumination causes infrared absorption changes linked to formation of the light-activated rhodopsin-transducin complex. In addition to the spectral changes normally associated with meta II formation, prominent absorption increases occur at 1735 cm-1, 1640 cm-1, 1550 cm-1, and 1517 cm-1. The D2O sensitivity of the broad carbonyl stretching band around 1735 cm-1 indicates that a carboxylic acid group becomes protonated upon formation of the activated complex. Reconstitution of rhodopsin into phosphatidylcholine vesicles has little influence on the spectral properties of the rhodopsin-transducin complex, whereas pH affects the intensity of the carbonyl stretching band. AC-terminal peptide comprising amino acids 340-350 of the transducin alpha-subunit reproduces the frequencies and isotope sensitivities of several of the transducin-induced bands between 1500 and 1800 cm-1, whereas an N-terminal peptide (aa 8-23) does not. Therefore, the transducin-induced absorption changes can be ascribed mainly to an interaction between the transducin-alpha C-terminus and rhodopsin. The 1735 cm-1 vibration is also seen in the complex with C-terminal peptides devoid of free carboxylic acid groups, indicating that the corresponding carbonyl group is located on rhodopsin.     

Opsin/all-trans-retinal complex activates transducin by different mechanisms than photolyzed rhodopsin

In rhodopsin, the 11-cis-retinal chromophore forms a complex with Lys296 of opsin via a protonated Schiff base. Absorption of light initiates the activation of rhodopsin by cis/trans photoisomerization of retinal. Thermal relaxation through different intermediates leads into the metarhodopsin states which bind and activate transducin (Gt) and rhodopsin kinase (RK). all-trans-Retinal also recombines with opsin independent of light, forming activating species of the receptor. In this study, we examined the mechanism by which all-trans-retinal activates opsin. To exclude other amines except active site Lys296 from formation of Schiff bases, we reductively methylated rhodopsin (PM-rhodopsin), which we then bleached to generate PM-opsin. Using spectroscopic methods and a Gt activation assay, we found that all-trans-retinal interacted with PM-opsin, producing a noncovalent complex that activated Gt. The residual nucleotide exchange in Gt catalyzed by opsin was approximately 1/250 lower relative to that of photoactivated rhodopsin (pH 8.0, 23 degrees C). Addition of equimolar all-trans-retinal led to an occupancy of one-tenth of the putative retinal binding site(s) of opsin and enhanced the Gt activation rate 2-fold. When the concentration of all-trans-retinal was increased to saturation, the Gt activation rate of the opsin/all-trans-retinal complex was approximately 1/33 lower compared to that of photoactivated rhodopsin. We conclude that all-trans-retinal can form a noncovalent complex with opsin that activates Gt by different mechanisms than photolyzed rhodopsin.     

Characterization of the mutant visual pigment responsible for congenital night blindness: a biochemical and Fourier-transform infrared spectroscopy study

A mutation in the gene for the rod photoreceptor molecule rhodopsin causes congenital night blindness. The mutation results in a replacement of Gly90 by an aspartic acid residue. Two molecular mechanisms have been proposed to explain the physiology of affected rod cells. One involves constitutive activity of the G90D mutant opsin [Rao, V. R., Cohen, G. B., & Oprian, D. D. (1994) Nature 367, 639-642]. A second involves increased photoreceptor noise caused by thermal isomerization of the G90D pigment chromophore [Sieving, P. A., Richards, J. E., Naarendorp F., Bingham, E. L., Scott, K., & Alpern, M. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 880-884]. Based on existing models of rhodopsin and in vitro biochemical studies of site-directed mutants, it appears likely that Gly90 is in the immediate proximity of the Schiff base chromophore linkage. We have studied in detail the mutant pigments G90D and G90D/E113A using biochemical and Fourier-transform infrared (FTIR) spectroscopic methods. The photoproduct of mutant pigment G90D, which absorbs maximally at 468 nm and contains a protonated Schiff base linkage, can activate transducin. However, the active photoproduct decays rapidly to opsin and free all-trans-retinal. FTIR studies of mutant G90D show that the dark state of the pigment has several structural features of metarhodopsin II, the active form of rhodopsin. These include a protonated carboxylic acid group at position Glu113 and increased hydrogen-bond strength of Asp83. Additional results, which relate to the structure of the active G90D photoproduct, are also reported. Taken together, these results may be relevant to understanding the molecular mechanism of congenital night blindness caused by the G90D mutation in human rhodopsin.     

The hydrogen-bonding network of water molecules and the peptide backbone in the region connecting Asp83, Gly120, and Glu113 in bovine rhodopsin

Difference Fourier transform infrared spectra were recorded between mutants of rhodopsin and their batho products. The pigments studied were single and combined mutants of intramembrane residues of bovine rhodopsin: Asp83, Glu113, Gly120, Gly121, and Glu122. Previous studies [Nagata, T., Terakita, A., Kandori, H., Kojima, D., Shichida, Y., and Maeda, A. (1997) Biochemistry 36, 6164-6170] showed that one of the water molecules which undergoes structural changes in this process forms hydrogen bonds with Glu113 and the Schiff base, and that another water molecule is linked to this structure through the peptide backbone. The present results show that this water molecule is located at the place that is affected by the replacements of Asp83 and Gly120 but only slightly by Gly121 and not at all by Glu122. Asp83 and Gly120 are close to each other, in view of the observations that the carboxylic C=O stretching vibration of Asp83 is affected by the G120A replacement and that each replacement affects the common peptide carbonyl groups. Our results suggest that these residues in the middle of helices B and C are linked-through a hydrogen-bonding network composed of water and the peptide backbone-with the region around Glu113.     

Development of an anti-bleeding agent for recombinant hirudin induced skin bleeding in the pig

A key step in visual transduction is the light-induced conformational changes of rhodopsin that lead to binding and activation of the G-protein transducin. In order to explore the nature of these conformational changes, time-resolved Fourier transform infrared spectroscopy was used to measure the kinetics of hydrogen/deuterium exchange in rhodopsin upon photoexcitation. The extent of hydrogen/deuterium exchange of backbone peptide groups can be monitored by measuring the integrated intensity of the amide II and amide II' bands. When rhodopsin films are exposed to D2O in the dark for long periods, the amide II band retains at least 60% of its integrated intensity, reflecting a core of backbone peptide groups that are resistant to H/D exchange. Upon photoactivation, rhodopsin in the presence of D2O exhibits a new phase of H/D exchange which at 10 degrees C consists of fast (time constant approximately 30 min) and slow (approximately 11 h) components. These results indicate that photoactivation causes buried portions of the rhodopsin backbone structure to become more accessible.     

Transmembrane signaling mediated by water in bovine rhodopsin

Unhydrated air-dried films of rhodopsin from bovine rod outer segment membranes do not produce its active state, metarhodopsin II. In order to reveal requirements for its formation, we studied changes in H-bonding of water, peptide carbonyl and carboxylic acid in the photochemical reactions by means of difference Fourier transform infrared spectroscopy, under both hydrated and unhydrated conditions. A water molecule near Glu113, which undergoes H-bonding change in bathorhodopsin, remained in the unhydrated film, but with a weaker H-bonding state than in the hydrated film. The other water molecules, which shfit in lumirhodopsin and metarhodopsin I as well as in bathorhodopsin of the hydrated film, were not observed in the unhydrated film. Effects of the dehydration were detected in all the C=O stretching vibrations of the peptide backbone and of Asp83 in the formation of bathorhodopsin. The C=O stretching band of Asp83 of lumirhodopsin and metarhodopsin I is intensified in the unhydrated film. We propose that structural changes at the intradiscal site in the interaction between the Schiff base and Glu113 affect water molecules, the peptide backbone, Asp83 and Glu122 in helices B and C through consecutive photochemical processes to metarhodopsin II.     

Hydration of the counterion of the Schiff base in the chloride-transporting mutant of bacteriorhodopsin: FTIR and FT-raman studies of the effects of anion binding when Asp85 is replaced with a neutral residue

The chromophores of the D85T and D85N mutants of bacteriorhodopsin are blue but become purple like the wild type when chloride or bromide binds near the Schiff base. In D85T this occurs near neutral pH, but in D85N only at pH < 4. The structures of the L and the unphotolyzed states of these proteins were examined with Fourier transform infrared spectroscopy. The difference spectra of the purple forms, but not the blue forms in the absence of these anions, resembled the spectrum of the wild-type protein. Shift of the ethylenic band toward lower frequency upon replacing chloride by bromide confirmed the contribution of the negative charge of the anions to the Schiff base counterion. These anions restored the change of water, which is bound near the protonated Schiff base but is absent in the blue form of the D85N mutant, though with stronger H-bonding than in the wild type. The C = N stretching vibration of the Schiff base in H2O and 2H2O was detected by Fourier transform Raman spectroscopy. The H-bonding strength of the Schiff base in the unphotolyzed state was weaker when chloride or bromide was bound to the mutants than with Asp85 as the counterion in the wild type. Thus, although the geometry of the environment is different, there is at least one water molecule coordinated to the bound halide in these mutants, in a way similar to water bound to Asp85 in the wild type.     

Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin: expression of 15N-lysine- and 13C-glycine-labeled opsin in a stable cell line

The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6-15N-lysine and 2-13C-glycine. Typical yields were 1.5-1.8 mg/liter. Incorporation of 6-15N-lysine was quantitative, whereas that of 2-13C-glycine was about 60%. The rhodopsin pigment formed by binding of 11-cis retinal was spectrally indistinguishable from native bovine rhodopsin. Magic angle spinning (MAS) NMR spectra of labeled rhodopsin were obtained after its incorporation into liposomes. The 15N resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-15N-lysine-labeled rhodopsin. This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 A, consistent with structural water in the binding site hydrogen bonded with the Schiff base nitrogen and the Glu-113 counterion. The present study demonstrates that structural studies of rhodopsin and other G protein-coupled receptors by using MAS NMR are feasible.     

Conformational changes in a mammalian voltage-dependent potassium channel inactivation peptide

Fast inactivation is restored in inactivation deletion mutant voltage-gated potassium (Kv) channels by application of synthetic inactivation 'ball' peptide. Using Fourier transform infrared and circular dichroism spectroscopy, we have investigated the structure of synthetic Kv3.4 channel ball peptide, in a range of environments relevant to the function of the ball domain. The ball peptide contains no alpha-helix or beta-sheet in reducing conditions in aqueous solution, but when cosolubilized with anionic lipid or detergent in order to mimic the environment which the ball domain encounters during channel inactivation, the ball peptide adopts a partial beta-sheet structure. Oxidation of the Kv3.4 ball peptide facilitates formation of a disulfide bond between Cys6 and Cys24 and adoption of a partial beta-sheet structure in aqueous solution; the tendency of the oxidized ball peptide to adopt beta-sheet is generally greater than that of the reduced ball peptide in a given environment. THREADER modeling of the Kv3.4 ball peptide structure predicts a beta-hairpin-like conformation which corresponds well to the structure suggested by spectroscopic analysis of the ball peptide in its cyclic arrangement. A V7E mutant Kv3.4 ball peptide analogue of the noninactivating Shaker B L7E mutant ball peptide cannot adopt beta-structure whatever the environment, and regardless of oxidation state. The results suggest that the Kv3.4 ball domain undergoes a conformational change during channel inactivation and may implicate a novel regulatory role for intramolecular disulfide bond formation in the Kv3.4 ball domain in vivo.     

Mechanisms of opsin activation

Rhodopsin is constrained in an inactive conformation by interactions with 11-cis-retinal including formation of a protonated Schiff base with Lys296. Upon photoisomerization, major structural rearrangements that involve protonation of the active site Glu113 and cytoplasmic acidic residues, including Glu134, lead to the formation of the active form of the receptor, metarhodopsin II b, which decays to opsin. However, an activated receptor may be generated without illumination by addition of all-trans-retinal or its analogues to opsin, as measured in this study by the increased phosphorylation of opsin by rhodopsin kinase. The potency of stimulation depended on the chemical and isomeric nature of the analogues and the length of the polyene chain with all-trans-C17 aldehyde and all-trans-retinal being the most active and trans-C12 aldehyde being the least active. Certain cis-isomers, 11-cis-13-demethyl-retinal and 9-cis-C17 aldehyde, were also active. Most of the retinal analogues tested did not regenerate a spectrally identifiable pigment, and many were incapable of Schiff base formation (ketone, stable oximes, and Schiff base-derivatives of retinal). Thus, receptor activation resulted from formation of non-covalent complexes with opsin. pH titrations suggested that an equilibrium exists between partially active (protonated) and inactive (deprotonated) forms of opsin. These findings are consistent with a model in which protonation of one or more cytoplasmic carboxyl groups of opsin is essential for activity. Upon addition of retinoids, the partially active conformation of opsin is converted to a more active intermediate similar to metarhodopsin II b. The model provides an understanding of the structural requirements for opsin activation and an interpretation of the observed activities of natural and experimental opsin mutants.     

Protein splicing in vitro with a semisynthetic two-component minimal intein

Protein splicing elements, or inteins, catalyze their own excision from flanking polypeptide sequences, or exteins, thereby leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. A trans-splicing system, using separately purified and expressed N- and C-terminal intein fragments of about 100 amino acids each, fused to appropriate exteins, was recently derived from the Mycobacterium tuberculosis RecA intein (Mills, K. V., Lew, B. M., Jiang, S.-Q., and Paulus, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3543-3548). We have replaced the C-terminal intein fragment of this system with synthetic peptides comprising 35-50 of the C-terminal residues of the RecA intein. The N-terminal intein fragment and the synthetic peptide were reconstituted by renaturation from guanidinium chloride. In the absence of added reductants, a disulfide-linked dimer of the N-terminal fragment and the peptide accumulated and could be induced to splice by reduction of its disulfide bond. The intermediate and spliced products were identified by polyacrylamide gel electrophoresis, mass spectrometry, and derivatization with thiol-reactive biotin followed by Western blotting with a streptavidin-enzyme conjugate. This is the first example of protein splicing involving a synthetic intein fragment and opens the way for studying the active site structure and function of the intein by the use of different synthetic peptides, including ones with non-natural amino acids.     

Chemical trapping of ternary complexes of human immunodeficiency virus type 1 integrase, divalent metal, and DNA substrates containing an abasic site. Implications for the role of lysine 136 in DNA binding

We report a novel assay for monitoring the DNA binding of human immunodeficiency virus type 1 (HIV-1) integrase and the effect of cofactors and inhibitors. The assay uses depurinated oligonucleotides that can form a Schiff base between the aldehydic abasic site and a nearby enzyme lysine epsilon-amino group which can subsequently be trapped by reduction with sodium borohydride. Chemically depurinated duplex substrates representing the U5 end of the HIV-1 DNA were initially used. We next substituted an enzymatically generated abasic site for each of 10 nucleotides normally present in a 21-mer duplex oligonucleotide representing the U5 end of the HIV-1 DNA. Using HIV-1, HIV-2, or simian immunodeficiency virus integrases, the amount of covalent enzyme-DNA complex trapped decreased as the abasic site was moved away from the conserved CA dinucleotide. The enzyme-DNA complexes formed in the presence of manganese were not reversed by subsequent addition of EDTA, indicating that the divalent metal required for integrase catalysis is tightly bound in a ternary enzyme-metal-DNA complex. Both the N- and C-terminal domains of integrase contributed to efficient DNA binding, and mutation of Lys-136 significantly reduced Schiff base formation, implicating this residue in viral DNA binding.     

Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA

Several mutations that cause severe forms of the human disease autosomal dominant retinitis pigmentosa cluster in the C-terminal region of rhodopsin. Recent studies have implicated the C-terminal domain of rhodopsin in its trafficking on specialized post-Golgi membranes to the rod outer segment of the photoreceptor cell. Here we used synthetic peptides as competitive inhibitors of rhodopsin trafficking in the frog retinal cell-free system to delineate the potential regulatory sequence within the C terminus of rhodopsin and model the effects of severe retinitis pigmentosa alleles on rhodopsin sorting. The rhodopsin C-terminal sequence QVS(A)PA is highly conserved among different species. Peptides that correspond to the C terminus of bovine (amino acids 324-348) and frog (amino acids 330-354) rhodopsin inhibited post-Golgi trafficking by 50% and 60%, respectively, and arrested newly synthesized rhodopsin in the trans-Golgi network. Peptides corresponding to the cytoplasmic loops of rhodopsin and other control peptides had no effect. When three naturally occurring mutations: Q344ter (lacking the last five amino acids QVAPA), V345M, and P347S were introduced into the frog C-terminal peptide, the inhibitory activity of the peptides was no longer detectable. These observations suggest that the amino acids QVS(A)PA comprise a signal that is recognized by specific factors in the trans-Golgi network. A lack of recognition of this sequence, because of mutations in the last five amino acids causing autosomal dominant retinitis pigmentosa, most likely results in abnormal post-Golgi membrane formation and in an aberrant subcellular localization of rhodopsin.     

Urbanization elicits a more atherogenic lipoprotein profile in carriers of the apolipoprotein A-IV-2 allele than in A-IV-1 homozygotes

PURPOSE: To test the effects of disruption of a conserved cysteine in the green cone opsin molecule on light-activated isomerization, transducin activation, folding, transport, and protein half-life. METHODS: Stable cell lines were established by transfecting 293-EBNA cells with a plasmid containing wild-type or mutant (C203R, C203S, C126S, C126S/C203S) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of cadmium chloride and analyzed by spectra, transducin activation, Western blotting, pulse-labeling with immunoprecipitation, and immunocytochemistry. RESULTS: The C203R mutation disrupts the folding and half-life of the green opsin molecule and its abilities to absorb light at the appropriate wavelength and to activate transducin. Similar disruption of folding, half-life, and light activation occurs when Cys203 or its presumed partner for formation of a disulfide bond (Cys126) is replaced by serine residues. CONCLUSIONS: Like rhodopsin, the folding of the cone opsins appears to be dependent on the formation of a disulfide bond between the third transmembrane helix and the second extracellular loop. Disruption of this disulfide bond represents a cause of color vision deficiencies that is unrelated to spectral shifts of the photopigment.     

Mechanisms of spectral tuning in blue cone visual pigments. Visible and raman spectroscopy of blue-shifted rhodopsin mutants

Spectral tuning by visual pigments involves the modulation of the physical properties of the chromophore (11-cis-retinal) by amino acid side chains that compose the chromophore-binding pocket. We identified 12 amino acid residues in the human blue cone pigment that might induce the required green-to-blue opsin shift. The simultaneous substitution of nine of these sites in rhodopsin (M86L, G90S, A117G, E122L, A124T, W265Y, A292S, A295S, and A299C) shifted the absorption maximum from 500 to 438 nm, accounting for 2,830 cm-1, or 80%, of the opsin shift between rhodopsin and the blue cone pigment. Raman spectroscopy of mutant pigments shows that the dielectric character and architecture of the chromophore-binding pocket are specifically altered. An increase in the number of dipolar side chains near the protonated Schiff base of retinal increases the ground-excited state energy gap via long range dipole-dipole Coulomb interaction. In addition, the W265Y substitution causes a decrease in solvent polarizability near the chromophore ring structure. Finally, two substitutions on transmembrane helix 3 (A117G and E122L) act in combination with the other substitutions to alter the binding-pocket structure, resulting in stronger interaction of the protonated Schiff base group with the surrounding dipolar groups and the counterion. Taken together, these results identify the amino acid side chains and the underlying physical mechanisms responsible for a majority of the opsin shift in blue visual pigments.     

NMR and molecular dynamics studies of tachykinins: conformation of the C-terminal pentapeptide of substance P(SP7-11)

Substance P belongs to the tachykinin family of neuropeptides which exhibit diverse pharmacological activity. The conformation of Phe1-Phe2-Gly3-Leu4-Met5-NH2 the C-terminal pentapeptide of substance P (SP7-11) has been studied by NMR and molecular dynamics (MD) methods. NMR studies were carried out both in DMSO-d6 and 95% H2O. Based on the observed chemical shifts, 3JNH alpha coupling constants, temperature coefficients of chemical shifts of NH resonances and the pattern of inter- and intraresidue NOE's, a predominantly extended backbone conformation has been deduced for the peptide in both DMSO and H2O. MD calculations carried out in vacuo indicate that the global minimum energy conformation of the molecule is folded with an intramolecular hydrogen bond between the protonated N-terminal and the C-terminal CONH2 group. The simulation shows that beta-turns are energetically unfavourable, while alpha-helices are seen to be unstable for the peptide. gamma-Bends at either Gly3 or Leu4 are the most preferred ones. Simulations carried out in DMSO as well as in water show a preference for a nearly extended conformation.     

Mechanism-based inactivation of a yeast methylamine oxidase mutant: implications for the functional role of the consensus sequence surrounding topaquinone

The copper-containing yeast methylamine oxidase E406N mutant has an altered consensus sequence surrounding the topaquinone cofactor (residue 405). The mutation has no effect on the final yield of the active-site topaquinone cofactor during biogenesis but causes the enzyme to be inactivated by substrate methylamine [Cai, D., and Klinman, J. P. (1994) Biochemistry 33, 7674-7653]. In this study we show that the inactivation leads to the formation of a covalent adduct, which has a UV/vis spectrum very similar to that of a product Schiff base, an intermediate of topaquinone-catalyzed amine oxidation reactions. The kinetic isotope effects on the second-order rate constant for the inactivation and catalytic turnover are identical, indicating that the two processes share a common intermediate that follows C_H bond cleavage. Resonance Raman spectroscopy provides direct evidence for the accumulation of a neutral product Schiff base species. Removal of excess methylamine leads to recovery of both activity and the native absorption spectrum for E406N, indicating that the cofactor in the inactivated enzyme is chemically competent for hydrolysis. The rate of the reactivation is slow, however; the shortest half-life of the inhibited E406N at 25 degrees C is 5.9 min at pH 6.15. pH effect experiments show that the inactivation and reactivation steps are controlled by a single ionizable group with a pKa of 6.9-7.1; under basic conditions, when this residue is deprotonated, the inactivation is the fastest and the half-life of the inhibited enzyme is the longest. On the basis of the available crystal structures of copper amine oxidases, we propose that a histidine residue in the dimer interface is responsible for the observed ionization. In the wild-type enzyme this histidine is kept protonated by virtue of Glu at position 406. Unlike methylamine, the larger substrates ethylamine and benzylamine give normal turnover with E406N. Disruption of structure at the subunit interface in E406N may allow a rotation of the relatively small topa-product Schiff base complex (formed from methylamine) away from the active-site base to a conformation that is incompetent toward hydrolysis.     

Transducin-alpha C-terminal peptide binding site consists of C-D and E-F loops of rhodopsin

The binding of heterotrimeric GTP-binding proteins (G-proteins) to serpentine receptors involves several independent contacts. We have deduced the points of interaction between mutant bovine rhodopsins and alphat-(340-350), a peptide corresponding to the C terminus of the alpha subunit (alphat) of bovine retinal G-protein, transducin. Direct binding of alphat-(340-350) to rhodopsin stabilizes the activated metarhodopsin II state (M II), consequently uncoupling the rhodopsin-transducin interaction. This peptide action requires two segments on the cytoplasmic domain of rhodopsin: the Tyr136-Val137-Val138-Val139 sequence on the C-D loop and the Glu247-Lys248-Glu249-Val250-Thr251 sequence on the E-F loop. We propose that a tertiary interaction of these two loop regions forms a pocket for binding the alphat C terminus of the transducin during light transduction in vivo. In most G-proteins, the C termini of alpha subunits are important for interaction with receptors, and, in several serpentine receptors, regions similar to those in rhodopsin are essential for G-protein activation, indicating that the interaction described here may be a generally applicable mode of G-protein binding in signal transduction.     

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.     

Identification of a common cytochrome P450 epitope near the conserved heme-binding petide with antibodies raised against recombinant cytochrome P450 family 2 proteins

The cytochrome P450 (P450) proteins constitute a superfamily of enzymes involved in various oxidations and related activities. Polyclonal antibodies raised against bacterial recombinant human P450s varied in specificity, depending upon the individual rabbits used. Several of the antisera raised against P450s 2C10 and 2E1 recognized a number of P450 family 1, 2, and 3 proteins, and two of the less selective antibodies were used to identify cross-reactive epitopes. P450 2C10 peptides reacting with anti-P450 2E1 and P450 2E1 peptides reacting with anti-P450 2C10 were isolated after electrophoresis/immunoblotting and analyzed by Edman degradation. Several of these were in a region near the highly conserved Cys that is a putative axial ligand to the heme. Peptides corresponding to the most conserved regions in this area were synthesized. Anti-P450 2C10 sera did not recognize 14-mer peptides corresponding to the heme-binding region (2C10 410-423 or 2E1 409-422) or the 14-mer peptides immediately C-terminal to these (2C10 425-438 or 2E1 424-437), but anti-P450 2E1 sera showed weak reaction with the latter two synthetic peptides. A longer peptide (29-mer) of P450 2E1 containing parts of both regions (412-440) reacted with both anti-P450 2C10 and anti-P450 2E1 antisera. Antibodies raised against a conjugate of the 29-mer peptide (with hemocyanin) recognized this antigen, the more C-terminal 14-mer peptides (2C10 425-438 and 2E1 424-437), P450s 2C10 and 2E1, and P450s 1A1, 11A1, and 17A. The 29-mer peptide showed considerable alpha-helix structure as judged by CD spectroscopy, in contrast to any of the 14-mers.(ABSTRACT TRUNCATED AT 250 WORDS)