Some Characteristics of Hydrolysis of Synthetic Substrates and Proteins by the Alkaline Proteinase from Aspergillus sojae

The specificity of the alkaline proteinase from Aspergillus sojae was investigated. In the specificity studies with synthetic substrates, the enzyme hydrolyzed the peptide linkages involving the carboxyl group of leucine, tyrosine, phenylalanine, arginine and lysine. In the hydrolysis of natural proteins, the enzyme liberated relatively large peptides and traces of free amino acids, suggesting that the enzyme is of a typical endo-type.

N- and C-Terminal amino acid residues appearing during time course digestion of various proteins were determined. Considering the influence of amino acid composition of substrates on the frequencies of appearance of the terminal amino acids, it was estimated that the susceptibility of peptide bonds of substrate to the enzyme depends mainly on the carboxyl side residues, and, to far less extent, on the amino side residues of the peptide bonds. The enzyme showed relatively high specificity for lysine, tyrosine, histidine, arginine and phenylalanine residues at the carboxyl side of the susceptible linkages.     

Isolation and properties of a capillary injury-related protease from lung lymph

Lung microvascular injury induced in sheep by intravenous infusion of Escherichia coli endotoxin, oleic acid, or air emboli caused the appearance in lung lymph of high levels of a protease with trypsin-like activity. The enzyme was isolated as an apparently homogeneous protein from pooled samples of active lung lymph, after an almost 9000-fold purification by affinity chromatography on columns of Reactive Blue 2-agarose, aprotinin-agarose, and p-aminobenzamidine-agarose, and chromatography on a column of Sephadex G-100. A molecular weight of about 70,000 to 75,000 was determined from mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The pH optimum was between 7.3 and 7.6. The isolated enzyme was quite labile, rapidly losing activity at both 37 and 25 degrees C. Addition of albumin to enzyme solutions protected against inactivation. Inhibition by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride indicated that the enzyme belongs to the class of serine proteases. The enzyme cleaved peptide bonds on the carboxyl side of arginine residues and showed a relatively high affinity toward peptides containing several basic amino acid residues. Bonds involving the carboxyl group of lysine were cleaved at a much slower rate. The enzyme showed no plasminogen activator activity and its substrate specificity was quite different from that of several proteases of the clotting cascade. Its appearance in lymph was not influenced by lymph clotting and the isolated enzyme was not capable of correcting the clotting defect of plasmas deficient in factors XII, XI, IX, VII, and X.     

Sites of covalent modification in Trg, a sensory transducer of Escherichia coli

The Trg protein mediates chemotactic response of Escherichia coli to the attractants ribose and galactose. Like other transducers, Trg is a transmembrane protein that undergoes post-translational covalent modification. The modifications are hydrolysis (deamidation) of certain glutamine side chains to create glutamate residues and methylation of specific glutamates to form carboxyl methyl esters. Analysis of radiolabeled, tryptic peptides by high performance liquid chromatography and gas-phase sequencing allowed direct identification of the modified residues of Trg. The protein has 5 methyl-accepting residues. Four, at positions 304, 310, 311, and 318, are contained in a 23-residue tryptic peptide ending in lysine. The fifth, at position 500, is within a 25-residue tryptic peptide ending in arginine. At two sites, 311 and 318, glutamines are deamidated to create methyl-accepting glutamates. There is not a required order of modification among the sites. However, there is a substantial preference for methylation on the arginine peptide and, among sites on the lysine peptide, for the middle pair. Comparison of sequences surrounding modified residues identified in this work for Trg and previously for Tsr and Tar suggests a consensus sequence for methyl-accepting sites of Ala/Ser-Xaa-Xaa-Glu-Glu*-Xaa-Ala/OH-Ala-OH/Ala, where OH signifies Ser or Thr and the asterick marks the site of modification.     

Specific cleavage of glucosephosphate isomerases at cysteinyl residues using 2-nitro-5-thiocyanobenzoic acid: analyses of peptides eluted from polyacrylamide gels and localization of active site histidyl and lysyl residues

Specific chemical cleavage of human placental and porcine muscle glucosephosphate isomerases at three amino peptide bonds of cysteinyl residues with 2-nitro-5-thiocyanobenzoic acid was achieved. Four primary peptides were generated from the cyanylated human glucosephosphate isomerase, indicating the quantitative cleavage of this enzyme. Four primary plus six overlap peptides were obtained from the cleavage of the swine muscle enzyme. The peptides were separated by SDS-polyacrylamide gel electrophoresis and eluted from the gels. Amino acid and carboxyl terminal analyses of the eluted peptides have permitted the alignment of these peptides with respect to the native polypeptide chain. The analysis of the enzyme which had been specifically covalently labeled at the essential lysine and histidine residues of the active center revealed that the active-site histidine and lysine residues are located on two distinct peptides with molecular weights of 27,500 and 14,000, respectively.     

Application of chemical selective cleavage methods to analyze post-translational modification in proteins

Three chemical specific cleavage reactions, one for the carboxyl side of aspartyl peptide bonds, one for the carboxyl side of asparaginyl peptide bonds and another for the amino side of seryl/threonyl peptide bonds have been recently established. Additionally, these reactions simultaneously react on several post-translationally modified groups in peptides or proteins. The modified groups cover the external modifications N-formyl, N-acetyl, N-pyroglutamyi residues and C-terminal-alpha amide, as well as the internal modifications such as O-acetyl serine, phosphorylated serine/tyrosine, sulfonylated tyrosine, glycosylated serine/threonine and glycosylated asparagine. These three cleavage reactions relate to key amino acids for modifications, deamidation for asparagine, phosphorylation and acetylation for serine, and glycosylation for asparagine, serine and threonine. The chemical reactions on these modifications change the peptide mapping pattern, and information from these reactions may contribute characterization and location of post-translational modified groups in the protein.     

Folding simulations of alanine-based peptides with lysine residues.

The folding of short alanine-based peptides with different numbers of lysine residues is simulated at constant temperature (274 K) using the rigid-element Monte Carlo method. The solvent-referenced potential has prevented the multiple-minima problem in helix folding. From various initial structures, the peptides with three lysine residues fold into helix-dominated conformations with the calculated average helicity in the range of 60-80%. The peptide with six lysine residues shows only 8-14% helicity. These results agree well with experimental observations. The intramolecular electrostatic interaction of the charged lysine side chains and their electrostatic hydration destabilize the helical conformations of the peptide with six lysine residues, whereas these effects on the peptides with three lysine residues are small. The simulations provide insight into the helix-folding mechanism, including the beta-bend intermediate in helix initiation, the (i, i + 3) hydrogen bonds, the asymmetrical helix propagation, and the asymmetrical helicities in the N- and C-terminal regions. These findings are consistent with previous studies.     

Mass spectrometric identification of the trypsin cleavage pathway in lysyl-proline containing oligotuftsin peptides.

Trypsin cleaves specifically peptide bonds at the C-terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4-NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ss-amyloid(4-10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys-N(epsilon)-side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P'2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions.     

Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminai OmpA signal sequence was fused with TEM β-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the β-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor, in the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal β-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the aminoterminal nuclease domain across the membrane, leaving the carboxyl terminal β-actamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.     

A Novel Type of Substrate Specificity of Rice BAPAase (Benzoyl-L-arginine p-nitroanilide Hydrolase) with Mixed Endopeptidase and Carboxypeptidase

The substrate specificity of rice embryo benzoyl-L-argininep-nitroanilide hydrolase (BAPAase) was examined. No endopeptidaseactivity toward protein substrates was detectable. Small peptides(less than 8 residues) and amide, ester substrates, however,were hydrolyzed very well at the carboxyl side of the lysineor arginine residue. No other peptide bond was hydrolyzed. TheN-terminal arginine of the substrates was released very slowly.Peptides with lysine or arginine penultimate to the C-terminalposition were hydrolyzed well and released an amino acid. Theoxidized insulin B chain (30 residues) was cleaved very slowlyat the C-terminal Lys-Ala bond, whereas an Arg-Gly bond at aninner position was not cleaved. The hydrolytic rate increasedafter the chain length was shortened by chymotryptic digestion.These results show that the rice embryo BAPAase is a novel enzymewhich has mixed endopeptidase-carboxypeptidase activity towardthe Arg-X and Lys-X bonds of small peptides, a characteristicintermediate between trypsin and serine carboxypeptidase. Thisenzyme may act in the breakdown of small peptides that havephysiological functions. (Received May 26, 1984; Accepted August 29, 1984)     

Purification and identification of a novel peptide derived from by-products fermentation of spiny head croaker (Collichthys lucidus) with antifungal effects on phytopathogens

In our previous study, spiny head croaker (Collichthys lucidus) by-products fermentation (SBF) products demonstrated plant growth promoting effects. In this study, the antimicrobial activities of SBF against plant pathogenic fungi and food-borne bacteria were evaluated. An active antifungal fraction SBF-3-1 was isolated from SBF using size exclusion chromatography and high performance liquid chromatography. Seven short peptides, i.e., LDEGW, DSFDFK, LDGW, FPDL, YNLDFK, FDGF and LDFE, plus one 22 residues peptide TFNTPAMYVAIQAVLSLYASGR were identified in SBF-3-1 by liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI-Q-TOF-MS/MS). The peptide TFNTPAMYVAIQAVLSLYASGR (named as SBF-3-1p), a potential antimicrobial peptide predicted by the antimicrobial peptide database (APD, http://aps.unmc.edu/AP/main.php), was further characterized. The prediction showed that the generation of SBF-3-1p could be associated with the action of trypsin and glutamyl endopeptidase, two proteases with specific cleavage sites on the carboxyl side of arginine (R) and lysine (K), and on the carboxyl side of Glu (E), respectively. According to the structural modeling, SBF-3-1p contained one α-helix and two loop regions. The synthesized SBF-3-1p exhibited strong activity against plant pathogenic fungus Pestalotiopsis sp. Therefore, SBF should be a good source of antifungal peptide, and could be used as natural fungicides in agriculture.     

Substrate recognition of isopeptidase: specific cleavage of the epsilon-(alpha-glycyl)lysine linkage in ubiquitin-protein conjugates

The structural chromatin protein A24 (uH2A) is a conjugate of histone H2A and a non-histone protein, ubiquitin. Eukaryotic cells contain an enzyme, generically termed isopeptidase, which can cleave A24 stoichiometrically into H2A and ubiquitin in vitro. Isopeptidase, free of proteinase activity, has been partially purified from calf thymus by ion-exchange chromatography, gel filtration and affinity chromatography, and analyzed for its substate specificity. There are three major types of isopeptide bonds besides the epsilon-(alpha-glycyl)lysine bond between H2A and ubiquitin; namely, the disulfide bridge, the aldol and aldimide bonds and the epsilon-(gamma-glutamyl)lysine crosslink. Under conditions where A24 was completely cleaved into H2A and ubiquitin, none of these naturally occurring isopeptide bonds was cleaved by isopeptidase. Furthermore, the bonds formed in vitro by transglutaminase reaction between casein and putrescine, through the gamma-NH2 of glutamine residue and the NH2 of putrescine, were not cleaved by the enzyme. The enzyme also failed to cleave the glycyl-lysyl and other orthodox peptide linkages within proteins. Among various proteins examined, the substrates for isopeptidase reaction were confined to conjugates between ubiquitin and other proteins, formed through epsilon-(alpha-glycyl)lysine bonds. Since ubiquitin released by isopeptidase is re-usable for an ATP-dependent conjugation with other proteins, its carboxyl terminal -Gly-Gly-COOH most likely is preserved intact, and is not blocked. These results suggest that isopeptidase specifically recognizes and cleaves the epsilon-(alpha-glycyl)lysine bond. A possible biological significance of this enzyme is discussed.     

Structural implications of placing cationic residues at either the NH2- or COOH-terminus in a pore-forming synthetic peptide

Restoration of chloride conductance via introduction of an anion-selective pore, formed by a channel-forming peptide, has been hypothesized as a novel treatment modality for patients with cystic fibrosis. Delivery of these peptides from an aqueous environment in the absence of organic solvents is paramount. M2GlyR peptides, designed based on the glycine receptor, insert into lipid bilayers and polarized epithelial cells and assemble spontaneously into chloride-conducting pores. Addition of 4 lysine residues to either terminus increases the solubility of M2GlyR peptides. Both orientations of the helix within the membrane form an anion-selective pore, however, differences in solubility, associations and channel-forming activity are observed. To determine how the positioning of the lysine residues affects these properties, structural characteristics of the lysyl-modified peptides were explored utilizing chemical cross-linking, NMR and molecular modeling. Initial model structures of the a-helical peptides predict that lysine residues at the COOH-terminus form a capping structure by folding back to form hydrogen bonds with backbone carbonyl groups and hydroxyl side chains of residues in the helical segment of the peptide. In contrast, lysine residues at the NH2-terminus form fewer H-bonds and extend away from the helical backbone. Results from NMR and chemical cross-linking support the model structures. The C-cap formed by H-bonding of lysine residues is likely to account for the different biophysical properties observed between NH2- and COOH-terminal-modified M2GlyR peptides.     

A study of hydrolysis of various synthetic peptides with gastrointestinal enzymes using thin-layer chromatography

Hydrolysis in vitro of alpha- and epsilon-peptide bonds of synthetic amino acids and peptide substrates,--models of protein fragments, with digestive enzymes was studied. The kinetics of hydrolysis was studied by quantitative thin-layer chromatography followed by densitometric analysis of the chromatographic patterns. The rate constants of hydrolysis of Phe-Lys, Gly-Lys dipeptides and their epsilon-acetyl and epsilon-succinyl derivatives with leucine aminopeptidase and pancreatic enzymes were calculated. epsilon-Acyl residues of the substrates failed to split off under these conditions. The digestive enzymes hydrolysed the alpha-peptide bonds adjacent to the acylated lysine. Hydrolysis of epsilon-acetyl substrates proceeded faster as compared to epsilon-succinyl derivatives.     

Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis

Endoproteinase Lys-C from Lysobacter enzymogenes, which is commercially available, proved to be useful in the determination of primary structures of proteins. The enzyme preferentially cleaves at the carboxyl side of lysine residues.     

A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.     

Dicyclohexylcarbodiimide cross-links two conserved residues, Asp-184 and Lys-72, at the active site of the catalytic subunit of cAMP-dependent protein kinase

In the absence of MgATP, the catalytic subunit of cAMP-dependent protein kinase is irreversibly inhibited by the hydrophobic carbodiimide dicyclohexylcarbodiimide, and this inhibition is most likely due to the formation of a cross-link between a carboxyl group and a lysine residue in the active site (Toner-Webb & Taylor, 1987). In order to identify these cross-linked residues, the catalytic subunit was modified by dicyclohexylcarbodiimide and then treated with acetic anhydride and digested with trypsin. The resulting peptides were resolved by high-performance liquid chromatography. One major absorbing tryptic peptide and one smaller peptide consistently and reproducibly showed a decrease in absorbance after the catalytic subunit had been treated with DCCD. These peptides correspond to residues 166-190 and 57-93, respectively. A unique peptide was isolated from the modified catalytic subunit, and the sequence of this peptide established that the cross-linking occurred between Asp-184 and Lys-72. The cross-linking of these two residues, which were both identified previously as essential residues, confirms the likelihood that each plays a role in the functioning of this enzyme. The fact that Asp-184 and Lys-72 appear to be invariant in all protein kinases further supports the hypothesis that these two residues, located close to one another at the active site of the enzyme, play essential roles in catalysis.     

The molecular basis of the temperature- and pH-induced conformational transitions in elastin-based peptides

Elastin undergoes an inverse temperature transition and collapses at high temperatures in both simulation and experiment. We investigated a pH-dependent modification of this transition by simulating a glutamic acid (Glu)-substituted elastin at varying pHs and temperatures. The Glu-substituted peptide collapsed at higher temperature than the unsubstituted elastin when Glu was charged. The charge effects could be reversed by neutralization of the Glu carboxyl groups at low pH, and in that case the peptide collapsed at a lower temperature. The collapse was accompanied by the formation of beta-turns and short distorted beta-sheets. Formation of contacts between hydrophobic side chains drives the collapse at high temperature, but interactions between water and polar groups (Glu and main chain) can attenuate this effect at high pH. The overall competition and balance of the polar and nonpolar groups determined the conformational states of the peptide. Water hydration contributed to the conformational transition, and the peptide and its hydration shell must be considered. Structurally, waters near polar residues mainly formed hydrogen bonds with the protein atoms, while waters around the hydrophobic side chains tended to be parallel to the peptide groups to maximize water-water interactions.     

Analysis of the peptidoglycan structure of Bacillus subtilis endospores.

Peptidoglycan was prepared from purified Bacillus subtilis spores of wild-type and several mutant strains. Digestion with muramidase resulted in cleavage of the glycosidic bonds adjacent to muramic acid replaced by peptide or alanine side chains but not the bonds adjacent to muramic lactam. Reduction of the resulting muropeptides allowed their separation by reversed-phase high-pressure liquid chromatography. The structures of 20 muropeptides were determined by amino acid and amino sugar analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In wild-type spores, 50% of the muramic acid had been converted to the lactam and 75% of these lactam residues were spaced regularly at every second muramic acid position in the glycan chains. Single L-alanine side chains were found on 25% of the muramic acid residues. The remaining 25% of the muramic acid had tetrapeptide or tripeptide side chains, and 11% of the diaminopimelic acid in these side chains was involved in peptide cross-links. Analysis of spore peptidoglycan produced by a number of mutants lacking proteins involved in cell wall metabolism revealed structural changes. The most significant changes were in the spores of a dacB mutant which lacks the sporulation-specific penicillin-binding protein 5*. In these spores, only 46% of the muramic acid was in the lactam form, 12% had L-alanine side chains, and 42% had peptide side chains containing diaminopimelic acid, 29% of which was involved in cross-links.     

Rat insulin-degrading enzyme: cleavage pattern of the natriuretic peptide hormones ANP, BNP, and CNP revealed by HPLC and mass spectrometry.

The degradation of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) by insulin-degrading enzyme (IDE) has been investigated. As revealed by high-performance liquid chromatography, all three peptides are sequentially cleaved at a limited number of sites, the latter of which were identified by mass spectrometric analyses. The studies revealed that ANP is preferred as substrate over BNP and CNP. ANP degradation is rapidly initiated by hydrolysis at the Ser25-Phe26 bond. Three additional cleavage sites were identified in ANP after prolonged incubation with IDE; in contrast, three and two bonds were hydrolyzed in BNP and CNP, respectively. Analysis of the nine cleavage sites shows a preference for basic or hydrophobic amino acid residues on the carboxyl side of a cleaved peptide bond. In contrast to most of the peptide fragments generated by IDE activity, the initial ANP cleavage product, F-R-Y, is rapidly degraded further by cleavage of the R-Y bond. Cross-linking studies with 125I-ANP in the presence of sulfhydryl-modifying agent indicate that IDE activity is inhibited at the level of initial substrate binding whereas metal-ion chelating agents only prevent hydrolysis. On the basis of its structural and enzymatic properties, IDE exhibits striking similarity to a number of recently-described endopeptidases.     

Electrostatic and hydrophobic forces tether the proximal region of the angiotensin II receptor (AT1A) carboxyl terminus to anionic lipids

The carboxyl terminus of the type-1 angiotensin II receptor (AT(1A)) is a focal point for receptor activation and deactivation. Synthetic peptides corresponding to the membrane-proximal, first 20 amino acids of the carboxyl terminus adopt an alpha-helical conformation in organic solvents, suggesting that the secondary structure of this region may be sensitive to hydrophobic environments. Using surface plasmon resonance, immobilized lipid chromatography, and circular dichroism, we examined whether this positively charged, amphipathic alpha-helical region of the AT(1A) receptor can interact with lipid components in the cell membrane and thereby modulate local receptor attachment and structure. A synthetic peptide corresponding to the proximal region of the AT(1A) receptor carboxyl terminus (Leu(305) to Lys(325)) was shown by surface plasmon resonance to bind with high affinity to the negatively charged lipid, dimyristoyl L-alpha-phosphatidyl-DL-glycerol (DMPG), but poorly to the zwitterionic lipid, dimyristoyl L-alpha-phosphatidylcholine (DMPC). In contrast, a peptide analogue possessing substitutions at four lysine residues (corresponding to Lys(307,308,310,311)) displayed poor association with either lipid, indicating a crucial anionic component to the interaction. Circular dichroism analysis revealed that both the wild-type and substituted peptides possessed alpha-helical propensity in methanol and trifluoroethanol, while the wild-type peptide also adopted partially inserted helical structure in DMPG and DMPC liposomes. In contrast, the substituted peptide exhibited spectra that suggested the presence of beta-sheet and alpha-helical structure in both liposomes. Immobilized lipid chromatography was used to characterize the hydrophobic component of the membrane interaction, and the results demonstrated that hydrophobic and electrostatic interactions mediated the binding of the wild-type peptide but that the substituted peptide bound to the model membranes mainly via hydrophobic forces. We propose that, in intact AT(1A) receptors, the proximal carboxyl terminus associates with the cytoplasmic face of the cell membrane via a high-affinity, anionic phospholipid-specific tethering that serves to increase the amphipathic helicity of this region. Such associations may be important for receptor function and common for G protein-coupled receptors.