Infrared spectroscopic study of photoreceptor membrane and purple membrane. Protein secondary structure and hydrogen deuterium exchange

Infrared spectroscopy in the interval from 1800 to 1300 cm-1 has been used to investigate the secondary structure and the hydrogen/deuterium exchange behavior of bacteriorhodopsin and bovine rhodopsin in their respective native membranes. The amide I' and amide II' regions from spectra of membrane suspensions in D2O were decomposed into constituent bands by use of a curve-fitting procedure. The amide I' bands could be fit with a minimum of three theoretical components having peak positions at 1664, 1638, and 1625 cm-1 for bacteriorhodopsin and 1657, 1639, and 1625 cm-1 for rhodopsin. For both of these membrane proteins, the amide I' spectrum suggests that alpha-helix is the predominant form of peptide chain secondary structure, but that a substantial amount of beta-sheet conformation is present as well. The shape of the amide I' band was pH-sensitive for photoreceptor membranes, but not for purple membrane, indicating that membrane-bound rhodopsin undergoes a conformation change at acidic pH. Peptide hydrogen exchange of bacteriorhodopsin and rhodopsin was monitored by observing the change in the ratio of integrated absorbance (Aamide II'/Aamide I') during the interval from 1.5 to 25 h after membranes were introduced into buffered D2O. The fraction of peptide groups in a very slowly exchanging secondary structure was estimated to be 0.71 for bacteriorhodopsin at pD 7. The corresponding fraction in vertebrate rhodopsin was estimated to be less than or equal to 0.60. These findings are discussed in relationship to previous studies of hydrogen exchange behavior and to structural models for both proteins.     

Albuminamide: a novel peptide amide isolated from bovine serum

During a systemic search for peptides that possess the C-terminal amide structure, a novel heptapeptide with isoleucine amide was isolated from bovine serum by sequential steps of reversed phase HPLC. Microsequence and amino acid analyses revealed the structure: Asp-Thr-His-Lys-Ser-Glu-Ile-NH2. Since this peptide has the identical sequence to N-terminal (1-7) fragment of bovine serum albumin (BSA), we have designated it albuminamide. The final HPLC step yielded 10 micrograms of homogeneous peptide preparation from 1 liter of bovine serum.     

Specific cleavages of arginyl peptide bonds at basic amino acid pairs by a serine proteinase from the microsomal membranes of rat liver

The specificity of action of a serine proteinase from the microsomal membranes of rat liver was investigated at pH 7.5 and 37 degrees C using various peptides as substrates. HPLC analyses of the peptides produced followed by their amino acid analyses have revealed that the enzyme is a unique endopeptidase specifically cleaving arginyl peptide bonds at paired basic amino acid residues. Thus, the enzyme is suggested to be a kind of processing proteinase involved in the conversion of proproteins to their mature forms. Indeed, the enzyme cleaved specifically the NH2-terminal 20-residue peptide of proalbumin at the Arg-Arg sequence.     

Polarized infrared spectroscopy of oriented purple membrane.

Polarized Fourier transform infrared spectroscopy has been used to study the structure of purple membrane from Halobacterium halobium. Membranes were oriented by drying a suspension of membrane fragments onto Irtran-4 slides. Dichroism measurements of the amide I, II and A peaks were used to find the average spatial orientation of the bacteriorhodopsin alpha-helices. By deriving a function that relates the observed dichroism to the orientational order parameters for the peptide groups, helical axis distribution, and mosaic spread of the membranes, the average orientation of the alpha-helices was found to lie in a range of less than 26 degrees away from the membrane normal, agreeing with electron microscopic measurements. The frequency of the amide I and A peaks is at least 10 cm-1 higher than values found for most alpha-helical polypeptides and proteins. This may indicate that bacteriorhodopsin contains distorted alpha-helical conformations.     

The disulfide structure of mouse lysosome-associated membrane protein 1

The disulfide structure of mouse lysosome-associated membrane protein 1 has been determined by reverse-phase isolation and sequence analysis of the cysteine-containing tryptic fragments of the reduced and non-reduced deglycosylated protein. Half-cystines were distinguished (a) by their localization within tryptic or chymotryptic peptides that formed reverse-phase peaks unique to the reduced digests and (b) by their 3H-carboxymethylation only after reduction of the protein. The disulfide arrangement of the cysteines was assigned after isolation of disulfide-linked peptide pairs. Each pair chromatographed as a peak present in the nonreduced (but not the corresponding reduced) tryptic digest. NH2-terminal sequencing as well as reduction, alkylation, and rechromatography of the disulfide-linked fragments led to the following assignment of disulfide bonds: Cys11 and Cys50, Cys125 and Cys161, Cys198 and Cys235, and Cys303 and Cys340. This structure creates four 36-38-residue loops that are symmetrically placed within the two halves of the protein's intraluminal domain. The loops formed by the Cys11-Cys50 and Cys198-Cys235 bridges are homologous, and the Cys125-Cys161 and Cys303-cys340 loops form a second set of homologous domains. The conservation of cysteine residues among lysosome-associated membrane proteins 1 and 2 suggests that this disulfide arrangement is common to both members of this family of lysosomal membrane glycoproteins.     

Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. I. Location of a group which is most reactive with N-ethylmaleimide

Ca2+-Transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum contains several SH groups which are reactive with N-ethylmaleimide (MalNEt) at pH 7.0. The location of the one which is most reactive with MalNEt (SHN, Kawakita et al. J. Biochem. 87, 609 (1980)) was identified on the amino acid sequence of the ATPase. SHN was labeled by reacting sarcoplasmic reticulum membranes with [14C] MalNEt to a labeling density of 1 mol/mol ATPase. [14C]MalNEt-labeled membranes were digested with thermolysin and 14C-labeled SHN peptides were fractionated by Sephadex LH-20 chromatography to give two major peaks of radioactivity. [14C]-MalNEt-labeled peptides were further purified to homogeneity by C18-reversed phase HPLC. Two radioactive peptides containing modified cysteine (Cys), Leu-Gly-Cys-Thr-Ser and Val-Cys-Lys-Met, were finally obtained in roughly equal amounts and in reasonable recovery. Both of these sequences were found in the amino acid sequence of Ca2+-transporting ATPase (Brandl et al. Cell 44, 597 (1986)), and Cys344 and Cys364 were identified as the targets of MalNEt-modification. Thus, 0.5 mol/mol ATPase of each Cys residue actually reacted rapidly with MalNEt under the conditions leading to SHN-modification. Modification of either one with MalNEt may negatively affect the reactivity of the other. Both of the highly reactive SH groups are located in the neighborhood of Asp351, the phosphorylation site of ATPase.     

Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65

The smaller isoform of the GABA synthesizing enzyme glutamic acid decarboxylase, GAD65, is synthesized as a soluble protein that undergoes post-translational modification(s) in the NH2-terminal region to become anchored to the membrane of small synaptic-like microvesicles in pancreatic beta cells, and synaptic vesicles in GABA-ergic neurons. A soluble hydrophilic form, a soluble hydrophobic form, and a hydrophobic firmly membrane-anchored form have been detected in beta cells. A reversible and hydroxylamine sensitive palmitoylation has been shown to distinguish the firmly membrane-anchored form from the soluble yet hydrophobic form, suggesting that palmitoylation of cysteines in the NH2-terminal region is involved in membrane anchoring. In this study we use site-directed mutagenesis to identify the first two cysteines in the NH2-terminal region, Cys 30 and Cys 45, as the sites of palmitoylation of the GAD65 molecule. Mutation of Cys 30 and Cys 45 to Ala results in a loss of palmitoylation but does not significantly alter membrane association of GAD65 in COS-7 cells. Deletion of the first 23 amino acids at the NH2 terminus of the GAD65 30/45A mutant also does not affect the hydrophobicity and membrane anchoring of the GAD65 protein. However, deletion of an additional eight amino acids at the NH2 terminus results in a protein which is hydrophilic and cytosolic. The results suggest that amino acids 24-31 are required for hydrophobic modification and/or targeting of GAD65 to membrane compartments, whereas palmitoylation of Cys 30 and Cys 45 may rather serve to orient or fold the protein at synaptic vesicle membranes.     

Action of peptidases in brain synaptic membranes on the NH2-terminus of adrenocorticotropin using ACTH-(1-16)-NH2 as a model substrate

The action of brain peptidases on NH2-terminal sequences of adrenocorticotropin was studied by incubation of ACTH-(1-16)-NH2 under different pH conditions. Profiles of metabolites and time course of product formation were obtained by HPLC analysis of the digests. Fragments of ACTH-(1-16)-NH2 were isolated and characterized by their amino acid composition and NH2-terminal groups. Both at pH 7.4 and pH 8.5 the following fragments were found: ACTH-(3-16)-NH2, ACTH-(4-16)-NH2, ACTH-(5-16)-NH2, and ACTH-(7-16)-NH2. At pH 7.4 the major products were ACTH-(4-16)-NH2 and ACTH-(7-16)-NH2, while the peptide ACTH-(3-16)-NH2 was the main metabolite at pH 8.5. The nature of identified peptides and the time course of their formation demonstrates that aminopeptidase activities predominate in the conversion of the NH2-terminus of adreno-corticotropin and related peptides by brain synaptic membranes.     

Amide III frequencies for Ala-X peptides depend on the X amino acid size

Raman spectra of series of aqueous solutions of peptides containing two amino acids, glycine-X, alanine-X, and serine-X, where X is an uncharged amino acid, show that the amide III band shifts systematically to lower frequencies as the side chain of the X amino acid becomes larger. The range of this shift is about 20 cm-1, starting at 1275 cm-1 for alanine-glycine and moving to 1251 cm-1 for alanine-tryptophan, with a correlation coefficient of 0.93 with the mass of the X amino acid side chain for 10 peptides. The amide I frequencies remain constant as the X amino acid is changed. This shift may result from a change in the average conformational preference of the peptide, a change in vibrational coupling of the amide III modes with the X amino acid side chain, a change in molecular force constants, or a combination of these. These results present a test for computational methods.     

Determination of solution structure and lipid micelle location of an engineered membrane peptide by using one NMR experiment and one sample

Antimicrobial peptides are universal host defense membrane-targeting molecules in a variety of life forms. Structure elucidation provides important insight into the mechanism of action. Here we present the three-dimensional structure of a membrane peptide in complex with dioctanoyl phosphatidylglycerol (D8PG) micelles determined by solution NMR spectroscopy. The model peptide, derived from the key antibacterial region of human LL-37, adopted an amphipathic helical structure based on 182 NOE-generated distance restraints and 34 chemical shift-derived angle restraints. Using the same NOESY experiment, it is also possible to delineate in detail the location of this peptide in lipid micelles via one-dimensional slice analysis of the intermolecular NOE cross peaks between the peptide and lipid. Hydrophobic aromatic side chains gave medium to strong NOE cross peaks, backbone amide protons and interfacial arginine side chain HN protons showed weak cross peaks, and arginine side chains on the hydrophilic face yielded no cross peaks with D8PG. Such a peptide-lipid intermolecular NOE pattern indicates a surface location of the amphipathic helix on the lipid micelle. In contrast, the epsilon HN protons of the three arginine side chains showed more or less similar intermolecular NOE cross peaks with lipid acyl chains when the helical structure was disrupted by selective d-amino acid incorporation, providing the basis for the selective toxic effect of the peptide against bacteria but not human cells. The differences in the intermolecular NOE patterns indicate that these peptides interact with model membranes in different mechanisms. Major NMR experiments for detecting protein-lipid NOE cross peaks are discussed.     

Controlled syntheses of natural and disulfide-mispaired regioisomers of alpha-conotoxin SI.

Methods are reported for the unambiguous syntheses of all three possible disulfide regioisomers with the sequence of alpha-conotoxin SI, a tridecapeptide amide from marine cone snail venom that binds selectively to the muscle subtype of nicotinic acetylcholine receptors. The naturally occurring peptide has two 'interlocking' disulfide bridges connecting Cys2-Cys7 and Cys3-Cys13 (2/7&3/13), while in the two mispaired isomers the disulfide bridges connect Cys2-Cys13 and Cys3-Cys7 (2/13 & 3/7, 'nested') and Cys2-Cys3 and Cys7-Cys13 (2/3 & 7/13, 'discrete'), respectively. Alignment of disulfide bridges was controlled at the level of orthogonal protection schemes for the linear precursors, assembled by Fmoc solid-phase peptide synthesis on acidolyzable tris(alkoxy)benzylamide (PAL) supports. Side-chain protection of cysteine was provided by suitable pairwise combination of the S-9H-xanthen-9-yl (Xan) and S-acetamidomethyl (Acm) protecting groups. The first disulfide bridge was formed from the corresponding bis(thiol) precursor obtained by selective deprotection of S-Xan, and the second disulfide bridge was formed by orthogonal co-oxidation of S-Acm groups on the remaining two Cys residues. It was possible to achieve the desired alignments with either order of loop formation (smaller loop before larger, or vice versa). The highest overall yields were obtained when both disulfides were formed in solution, while experiments where either the first or both bridges were formed while the peptide was on the solid support revealed lower overall yields and poorer selectivities towards the desired isomers.     

Determination of solution structure and lipid micelle location of an engineered membrane peptide by using one NMR experiment and one sample

Antimicrobial peptides are universal host defense membrane-targeting molecules in a variety of life forms. Structure elucidation provides important insight into the mechanism of action. Here we present the three-dimensional structure of a membrane peptide in complex with dioctanoyl phosphatidylglycerol (D8PG) micelles determined by solution NMR spectroscopy. The model peptide, derived from the key antibacterial region of human LL-37, adopted an amphipathic helical structure based on 182 NOE-generated distance restraints and 34 chemical shift-derived angle restraints. Using the same NOESY experiment, it is also possible to delineate in detail the location of this peptide in lipid micelles via one-dimensional slice analysis of the intermolecular NOE cross peaks between the peptide and lipid. Hydrophobic aromatic side chains gave medium to strong NOE cross peaks, backbone amide protons and interfacial arginine side chain H^N protons showed weak cross peaks, and arginine side chains on the hydrophilic face yielded no cross peaks with D8PG. Such a peptide-lipid intermolecular NOE pattern indicates a surface location of the amphipathic helix on the lipid micelle. In contrast, the εH^N protons of the three arginine side chains showed more or less similar intermolecular NOE cross peaks with lipid acyl chains when the helical structure was disrupted by selective d-amino acid incorporation, providing the basis for the selective toxic effect of the peptide against bacteria but not human cells. The differences in the intermolecular NOE patterns indicate that these peptides interact with model membranes in different mechanisms. Major NMR experiments for detecting protein-lipid NOE cross peaks are discussed.     

Amide hydrogen exchange rates of peptides in H2O solution by 1H nuclear magnetic resonance transfer of solvent saturation method. Conformations of oxytocin and lysine vasopressin in aqueous solution.

The NH exchange rates in aqueous media of oxytocin and 8-lysine vasopressin (LVP) have been measured by using transfer of solvent saturation method. The data are consistent with a "highly motile" dynamic equilibrium between folded and highly solvated conformations. The highly-motility limit applies to the exchange of NH hydrogens of oxytocin and LVP. Folded structures are more prevalent in oxytocin than in LVP. Partial shielding is indicated for peptide hydrogens of Asn5 and perhaps also Cys6 of oxytocin and for Cys6 of LVP. It is tentatively proposed that the folded conformation of oxytocin in aqueous media may contain a parallel beta-structure in the tocinamide ring consisting of two hydrogen bonds: one between the Tyr2 C = O and Asn5 peptide NH as originally proposed for the preferred conformation of oxytocin in dimethyl sulfoxide (D. W. Urry and R. Walter), and the second between he Cys1 C = O and the Cys6 NH. In LVP the hydrogen bond between the Tyr2 C = O and Asn5 peptide NH appears to be absent. The acylic tripeptide sequences (-Pro-X-Gly-NH2) of both hormones appear to be predominantly solvated. The second-order rate constants for acid catalyzed exchange of the primary amide hydrogens of Gln4, Asn5, and Gly9 of oxytocin are consistently greater for the trans NH than for the corresponding cis NH. This observation can be rationalized in terms of mechanisms involving protonation of either the amide oxygen, or the amide nitrogen, but with limited rotation about the C - N bond.     

Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH(2)-Gly(3) substrates

Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. We find that purified recombinant sortase hydrolyzed peptides bearing an LPXTG motif at the peptide bond between threonine and glycine. In the presence of NH(2)-Gly(3), sortase catalyzed exclusively a transpeptidation reaction, linking the carboxyl group of threonine to the amino group of NH(2)-Gly(3). In the presence of amino group donors the rate of sortase mediated cleavage at the LPXTG motif was increased. Hydrolysis and transpeptidation required the sulfhydryl of cysteine 184, suggesting that sortase catalyzed the transpeptidation reaction of surface protein anchoring via the formation of a thioester acyl-enzyme intermediate.     

Determination of the positions of the disulfide bonds in aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1

Aqualysin I is a heat-stable alkaline serine protease produced by Thermus aquaticus YT-1. Aqualysin I comprises 281 amino acid residues and contains four cysteine residues. The cysteine residues seemed to form disulfide bonds in the molecule. Thus, the positions of the disulfide bonds were investigated. Disulfide bond-containing peptides were identified by peptide mapping with HPLC before and after carboxymethylation of chymotryptic peptides of aqualysin I. The disulfide bond-containing peptides were isolated and then carboxymethylated. Carboxymethylcysteine-containing peptides were purified, and their amino acid compositions and sequences were determined. Based on the data obtained and the primary structure of aqualysin I, it was concluded that two disulfide bonds were formed between Cys67 and Cys99, and between Cys163 and Cys194.     

Studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin.

Influenza virus hemagglutinin (HA) fuses membranes at endosomal pH by a process which involves extrusion of the NH2-terminal region of HA2, the fusion peptide, from its buried location in the native trimer. We have examined the amino acid sequence requirements for a functional fusion peptide by determining the fusion capacities of site-specific mutant HAs expressed by using vaccinia virus recombinants and of synthetic peptide analogs of the mutant fusion peptides. The results indicate that for efficient fusion, alanine can to some extent substitute for the NH2-terminal glycine of the wild-type fusion peptide but that serine, histidine, leucine, isoleucine, or phenylalanine cannot. In addition, mutants containing shorter fusion peptides as a result of single amino acid deletions are inactive, as is a mutant containing an alanine instead of a glycine at HA2 residue 8. Substitution of the glycine at HA2 residue 4 with an alanine increases the pH of fusion, and valine-for-glutamate substitutions at HA2 residues 11 and 15 are without effect. We confirm previous reports on the need for specific HAo cleavage to generate functional HAs, and we show that both inappropriately cleaved HA and mutant HAs, irrespective of their fusion capacities, upon incubation at low pH undergo the structural transition required for fusion.     

Hydrogen kinetics of peptide amide protons at the bovine pancreatic trypsin inhibitor protein-solvent interface

Hydrogen exchange rate constants of the 25 most rapidly exchanging peptide amide protons in bovine pancreatic trypsin inhibitor have been determined over a range of pH that spans pH min, the pH of minimum rate. Most of these are on the protein surface, exposed to solvent and not hydrogen bonded in the crystal structure. Contrary to commonly held assumptions, the exchange kinetics of surface NH groups are not equivalent to the kinetics of NH groups in peptides in the extended configuration. All surface NH groups exchange more slowly than NH groups in model peptides, with rate constants distributed over a range of more than two orders of magnitude. In addition, their pH min values vary widely. For most of the surface NH groups, pH min is lower than in model compounds and, for several, pH min is less than 1. These results indicate that the local environment of the surface peptide groups when the exchange event occurs is very different from that of extended peptides. Analysis based on consideration of an O-protonation mechanism for acid catalysis and of electrostatic effects on exchange kinetics further indicates (see the accompanying paper) that, in general, exchange of surface NH groups occurs from a conformation of the protein approximated by the crystal structure. The 1H-2H exchange rate constants were measured from 300 MHz nuclear magnetic resonance spectra in which assigned surface N1H resonances are resolved by the use of partially deuterated protein samples. A marked pH dependence of the chemical shifts observed in the pH range 1 to 4.5 for several surface NH groups reflects the titration of nearby carboxyl groups.     

Conformation,orientation, and adsorption kinetics of dermaseptin B2 onto synthetic supports at aqueous/solid interface

The antimicrobial activity of cationic amphipathic peptides is due mainly to the adsorption of peptides onto target membranes, which can be modulated by such physicochemical parameters as charge and hydrophobicity. We investigated the structure of dermaseptin B2 (Drs B2) at the aqueous/synthetic solid support interface and its adsorption kinetics using attenuated total reflection Fourier transform infrared spectroscopy and surface plasmon resonance. We determined the conformation and affinity of Drs B2 adsorbed onto negatively charged (silica or dextran) and hydrophobic supports. Synthetic supports of differing hydrophobicity were obtained by modifying silica or gold with omega-functionalized alkylsilanes (bromo, vinyl, phenyl, methyl) or alkylthiols. The peptide molecules adsorbed onto negatively charged supports mostly had a beta-type conformation. In contrast, a monolayer of Drs B2, mainly in the alpha-helical conformation, was adsorbed irreversibly onto the hydrophobic synthetic supports. The conformational changes during formation of the adsorbed monolayer were monitored by two-dimensional Fourier transform infrared spectroscopy correlation; they showed the influence of peptide-peptide interactions on alpha-helix folding on the most hydrophobic support. The orientation of the alpha-helical Drs B2 with respect to the hydrophobic support was determined by polarized attenuated total reflection; it was around 15 +/- 5 degrees. This orientation was confirmed and illustrated by a molecular dynamics study. These combined data demonstrate that specific chemical environments influence the structure of Drs B2, which could explain the many functions of antimicrobial peptides.     

Hydrogen--deuterium exchange kinetics of the amide protons of oxytocin studied by nuclear magnetic resonance

The contribution of intramolecular hydrogen bonding to the solution structure of oxytocin was evaluated by study of amide hydrogen exchange rates in D2O by Fourier transform 1H NMR spectroscopy. Resolution enhancement filtering was employed in the determination of individual pseudo-first-order rate constants. Apparent barriers to exchange of 0.5 and 0.6 kcal mol-1 were measured for Asn5 and Cys6 peptide NH, respectively. The slowing is best explained by steric hindrance to solvent access in the case of Asn5, while for the Cys6 participation in a weak intramolecular hydrogen bond is possible. Fourfold acceleration of base-catalyzed exchange was observed for Tyr2 NH; it is proposed that this is the result of electronic effects induced by hydrogen bonding of Cys1 C=0, either to Cys6 NH or to the N-terminal amino group. Exchange proceeds near the random coil limit for each of the remaining residues. Comparison with exchange data for the model tripeptide N-acetyl-L-prolyl-L-leucylglycinamide demonstrates no evidence of noncovalent association of the tocin ring with the tripeptide tail of the hormone.