alpha-Chymotrypsin as the catalyst for peptide synthesis.

alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.     

Pepstatin-insenstive acid proteases from Scytalidium lignicolum. Kinetic study with synthetic peptides.

A kinetic study was conducted on the acid proteases A-1 and A-2 from Scytalidium lignicolum using synthetic peptides as substrates. Almost maximum activity was attained with N-acylated tetrapeptides as the molecular size of substrates was increased. Suitable amino acid residues were required at the P1-P2 and P1'-P2' positions [notation of Schechter and Berger (14)]. Hydrophobic or bulky residues such as leucine were specifically required at the P1 and P1' positions, with the specificity at the latter position being considerably lower than that at the former. For catalysis, the presence of certain amino acid residues at the P2 and P2' positions was essential, mainly in relation to kcat. An inhibition study supported this view. Stringent stereospecificity was observed at the P2 and P2' positions, but the side chain specificity was low. Study of the B enzyme from the same organism was very difficult owing to its low activity against the peptides used. The Scytalidium acid proteases A-1, A-2, and B showed considerably different behavior against peptide substrates in comparison with usual acid proteases, which are senstive to pepstatin.     

Automated design of the surface positions of protein helices.

Using a protein design algorithm that quantitatively considers side-chain interactions, the design of surface residues of alpha helices was examined. Three scoring functions were tested: a hydrogen-bond potential, a hydrogen-bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen-bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4-p1 were designed by using the Dead-End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 degree C, with melting temperatures from 69-72 degrees C, over 12 degrees C higher than GCN4-p1, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 degrees C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions.     

Amino acid determinants for NMDA receptor inhibition by conantokin-T

Several derivatives of conantokin-T (con-T), a naturally occurring, gamma-carboxyglutamate (Gla)-containing peptide with NMDA receptor (NMDAR) antagonist properties, were synthesized and evaluated for their ability to displace [(3)H]MK-801 from adult rat forebrain membranes. Analyses of progressive C-terminal truncation analogs of the parent 21-mer revealed gradual losses in activity with decreased chain length. In this series, con-T[1-8] was identified as the shortest variant capable of manifesting inhibitory activity (< 1% of the parent peptide). Ala substitution studies of individual residues identified Gly1, Gla3, Met8 and Leu12 as important for activity, while Glu2, Gla4 and Tyr5 were shown to be essential in this regard. The effect of side-chain length and charge in the N-terminal region was probed by single amino acid replacements. No correlation was observed between potencies and circular dichroism-derived helical contents of the con-T derivatives. Further elaboration of structure-function relationships in con-T was effected through the design and synthesis of helically constrained and destabilized analogs. The results of the current study were compared with those of a previous investigation on con-G, a related conantokin. Substantial differences in activity requirements were noted between the peptides, particularly in the C-terminal regions. Chimeras of con-T and con-G were generated and revealed virtually no interchangeability of residues between these two peptides. Finally, single amino acid substitutions that resulted in analogs with enhanced inhibitory properties were combined to yield superior conantokin-based NMDAR inhibitors.     

Specificity determinants of acylaminoacyl-peptide hydrolase.

In an attempt to explore how specific features of the substrate's primary structure may affect the activity of rabbit muscle acylaminoacyl-peptide hydrolase (EC 3.4.19.1), a number of acetylated peptides containing specific amino acid replacements in specific positions were prepared and compared as substrates for the hydrolase. The principal variants were D-Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effect of the length of the peptide was also investigated in a less systematic manner. The substrates were either prepared by direct acetylation of peptides, by extension of the N-terminus with acetylamino acids or acetylpeptides, activated as N-hydroxysuccinimide esters, or by isolation of the N-terminal peptides from naturally occurring acetylated proteins. It was found that D-Ala on either side of the bond to be cleaved (positions 1 and 2) completely inhibited the enzymatic activity, whereas acetylated peptides with D-Ala in positions 3 or 4 were as good substrates as those containing L-Ala. Peptides with Pro in positions 2 were also inactive, and most of the peptides with Pro in the third position were very poor substrates; only the peptide Ac-AAP gave reasonably high activity (30% of Ac-AAA), which was reduced to 1-2% if additional residues were present at the C-terminus (Ac-AAPA, Ac-AAPAA). The presence of a positive charge in positions 2, 3, 4, 5, and 6 gave strong reduction in hydrolase activity varying with the charge's distance from the N-terminus from 0 to 15-20% of the rates obtained with the reference peptides without positive charges.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of tyrosinase from Neurospora crassa. I. Purification and amino acid sequence of the cyanogen bromide fragments

Cyanogen bromide (CB) cleavage of Neurospora tyrosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionylthreonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein. The amino acid sequences of the cyanogen bromide fragments CB2, CB3, and CB4 were determined by a combination of automated and manual sequence analysis on peptides derived by chemical and enzymatic cleavage of the intact and the maleylated derivatives. The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids whose chemical structure will be presented in the following paper.     

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.     

Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha.

Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.     

Energetics and cooperativity of the hydrogen bonding and anchor interactions that bind peptides to MHC class II protein

The complexity of the interaction between major histocompatibility complex class II (MHC II) proteins and peptide ligands has been revealed through structural studies and crystallographic characterization. Peptides bind through side-chain "anchor" interactions with MHC II pockets and an extensive array of genetically conserved hydrogen bonds to the peptide backbone. Here we quantitatively investigate the kinetic hierarchy of these interactions. We present results detailing the impact of single side-chain mutations of peptide anchor residues on dissociation rates, utilizing two I-A(d)-restricted peptides, one of which has a known crystal structure, and 24 natural and non-natural amino acid mutant variants of these peptides. We find that the N-terminal P1, P4 and P6 anchor-pocket interactions can make significant contributions to binding stability. We also investigate the interactions of these peptides with four I-A(d) MHC II proteins, each mutated to disrupt conserved hydrogen bonds to the peptide backbone. These complexes exhibit kinetic behavior suggesting that binding energy is disproportionately invested near the peptide N terminus for backbone hydrogen bonds. We then evaluate the effects of simultaneously modifying both anchor and hydrogen bonding interactions. A quantitative analysis of 71 double mutant cycles reveals that there is little apparent cooperativity between anchor residue interactions and hydrogen bonds, even when they are directly adjacent (<5A).     

E.s.r. of spin-trapped radicals in aqueous solutions of peptides. Reactions of the hydroxyl radical.

The reactions of hydroxyl radicals with 30 dipeptides and several larger peptides were studied in aqueous solutions. The OH radicals were generated by U.V. photolysis of H2O2. The short-lived peptide radicals were spin-trapped using t-nitrosobutane and identified by e.s.r. For dipeptides containing the amino terminal residues glycine, alanine and phenylalanine, abstraction of the hydrogen from the carbon adjacent to the peptide nitrogen was the major process leading to the spin-adducts. Such radicals will be referred to as backbone radicals. Dipeptides with a carbonyl terminal serine residue and also glycylglutamic acid form both backbone and side-chain radicals, with the latter being formed in larger quantities. For dipeptides, side-chain radicals were detected on either the carboxyl or amino terminal residues of both. The effect of pD on the e.s.r. sectrum of the spin-adducts of glycylglycine was studied and the pK of the carboxyl group of this radical was determined to be 2.5. For (Ala)3 and (Ala)n, with an average value of n = 1800, backbone and minor side-chain radicals were observed. For ribonucleases-S-peptide, containing 20 amino acid residues, both backbone and side-chain radicals were detected.     

Tritiated photoactivatable analogs of the native human thrombin receptor (PAR-1) agonist peptide, SFLLRN-NH2.

Six photoactivatable analogs of the human thrombin receptor activating peptide (TRAP), SFLLRN-NH2, were synthesized by substituting the photoactive amino acid, p-benzoylphenylalanine (Bpa), into each position of the peptide sequence. Platelet aggregation assays indicated that the peptides with Bpa substitutions at positions 3 to 6 retained agonist activity. These peptides were prepared in tritiated form as potential thrombin receptor photoaffinity labels. The [3H]Bpa-containing analogs were constructed by resynthesizing the peptides with the amino acid, 4-benzoyl-2',5'-dibromophenylalanine (Br2Bpa), and subjecting the purified peptides to Pd-catalyzed tritiodebromination. The radiochemical yields for the reductive tritiation were < 2% for peptides with [3H]Bpa in the third and fourth positions, and between 7 and 16% for the peptides with substitutions at the fifth and sixth positions. The low yields were due to over-reduction of the Bpa carbonyl group and nonspecific degradation during reductive tritiation. This report describes the first use of Br2Bpa for the preparation of tritiated photoactivatable peptides.     

Side chain contributions to the interconversion of the topological isomers of guanylin-like peptides.

The peptide hormones guanylin and uroguanylin are ligands of the intestinal guanylyl cyclase-C (GC-C) that is involved in the regulation of epithelial water and electrolyte transport. The small peptides contain 15 and 16 amino acids, respectively, and two disulfide bonds with a 1-3/2-4 connectivity. This structural feature causes the unique existence of two topological isoforms for each peptide in an approximate 3:2 ratio, with only one of the isoforms exhibiting GC-C-activating potential. The two uroguanylin isomers can be separated by HPLC and are of sufficient stability to be studied separately at ambient temperatures while the two guanylin isomers are rapidly interconverting even at low temperatures. Both isomers show clearly distinguishable (1)H chemical shifts. To investigate the influence of certain amino acid side chains on this isomerism and interconversion kinetics, derivatives of guanylin and uroguanylin (L-alanine scan and chimeric peptides) were designed and synthesized by Fmoc solid-phase chemistry and compared by HPLC and 2D (1)H NMR spectroscopy. Amino acid residues with the most significant effects on the interconversion kinetics were predominantly identified in the COOH-terminal part of both peptides, whereas amino acids in the central part of the peptides only moderately affected the interconversion. Thus, the conformational conversion among the isomers of both peptides is under the control of a COOH-terminal sterical hindrance, providing a detailed model for this dynamic isomerism. Our results demonstrate that kinetic control of the interconversion process can be achieved by the introduction of side chains with a defined sterical profile at suitable sequence positions. This is of potential impact for the future development of GC-C peptide agonists and antagonists.     

DNA sequence-specific recognition of peptides incorporating the HPRK and polyamide motifs

Three peptide amides, HPRK(Py)(4)HPRK-NH(2) (PyH-12), HPRK(Py)(3)HPRK-NH(2) (PyH-11) and HPRK(Py)(2)HPRK-NH(2) (PyH-10), incorporating two HPRK motifs and various 4-amino-1-methylpyrrole-2-carboxylic acid residues (Py) were synthesized by solid-phase peptide methodology. The binding of these three peptides to a 5'-32P-labeled 158-mer DNA duplex (Watson fragment) and to a 5'-32P-labeled 135-mer DNA duplex (complementary Crick fragment) was investigated by quantitative DNase I footprinting. On the 158-mer Watson strand, the most distinctive DNase I blockages seen with all three peptides occur around positions 105-112 and 76-79, corresponding to the sequences 5'-GAGAAAAT-3' and 5'-CGGT-3', respectively. However, on the complementary Crick strand, only PyH-12 strongly discriminates the 5'-TTT-3' site around positions 108-110 whereas both PyH-11 and PyH-10 have moderate binding around positions 102-112 comprising the sequence 5'-ATTTTCTCCTT-3'. Possible bidentate and single interactions of the side-chain functions and alpha-amino protons of the peptides with DNA bases are discussed.     

Synthesis of N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine: its use in peptide synthesis for placing a bromoacetyl cross-linking function at any desired sequence position.

A new amino acid derivative, N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-beta-alanine with N alpha-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for N alpha-Boc amino acid derivatives. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers, also linked through thioether bonds. Products made with BBAL peptides may be characterized by amino acid analysis of acid hydrolyzates by quantification of beta-alanine, which separates from natural amino acids in suitable analytical systems. Where sulfhydryl groups on coupling partners arise from cysteine residues, S-(carboxymethyl)cysteine in acid hydrolyzates may also be assayed for this purpose. Examples are given of the use of BBAL in preparing peptide polymers and a peptide conjugate with bovine albumin to serve as immunogens or model vaccine components.     

Structure-activity relationships for the cardiotropic action of the Led-NPF-I peptide in the beetles Tenebrio molitor and Zophobas atratus.

We have examined the effects of the Led-NPF-I peptide (Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide) and a series of ten analogues on the heart contractile activity of Tenebrio molitor and Zophobas atratus, and the structure-activity relationships for cardioactive action of Led-NPF-I were established. A video microscopy technique and computer-based method of data acquisition and analysis were used to study the action of the peptides on continuously perfused heart preparations. Cardiac activity was progressively inhibited by Led-NPF-I when the peptide concentrations were increased from 10(-9) to 10(-5) M. Substitution of the L-proline residue at position 4 of the native peptide with hydroxyproline, valine or D-proline caused a loss of cardioinhibitory activity. Also, replacement of arginine residues at all three positions 2, 7 and 9 with another basic amino acid histidine, reduces cardioinhibitory action of Led-NPF-I. Some modifications of the C-terminal residues, as the Phe(4-NO2)-, Phe(4-NH2)- and Phe(4-NMe2)-analogues, resulted in agonistic peptides with biological activity similar to that of the native peptide. However, three other C-terminal analogues tested [Tyr10]-, [D-Phe10]-Led-NPF-I, and Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-OH were inactive in the heart bioassay, which suggests that this end of the amino acid chain may play an important role in bioactivity and interaction of the native peptide with its receptor on the myocardium.     

Vitamin K and the biosynthesis of prothrombin. V. Gamma-carboxyglutamic acids, the vitamin K-dependent structures in prothrombin.

Tryptic peptides obtained from normal prothrombin have been compared with those obtained from prothrombin synthesized by cattle given the vitamin K antagonist dicumarol. Two peptides were found which contain vitamin K-dependent structures. These peptides contain residues 4 through 10 and residues 12 through 44, respectively. One of these (residues 4 through 10) has previously been shown to contain gamma-carboxyglutamic acid residues. Digestion of this peptide with aminopeptidase M and carboxypeptidase B yielded a tetrapeptide (residues 6 through 9). Mass spectra of this peptide showed that it has the structure Leu-Glu(CO2)-Glu(CO2)-Val. The structure of the peptide containing residues 12 through 44 was determined by automated degradation in a peptide sequenator. The modified glutamic acid residues were identified by mass spectrometric comparison with the thiohydantoin derivatives of synthetic gamma-carboxyglutamic acid. This approach unequivocally demonstrated that all of the first 10 glutamic acid residues in prothrombin are carboxylated to form gamma-carboxyglutamic acid residues. Evidence is also presented that indicates that these gamma-carboxyglutamic acid residues constitute the entire vitamin K-dependent modification of prothrombin.     

Side-chain structures in the first turn of the alpha-helix

The first three residues at the N terminus of the alpha-helix are called N1, N2 and N3. We surveyed 2102 alpha-helix N termini in 298 high-resolution, non-homologous protein crystal structures for N1, N2 and N3 amino acid and side-chain rotamer propensities and hydrogen-bonding patterns. We find strong structural preferences that are unique to these sites. The rotamer distributions as a function of amino acid identity and position in the helix are often explained in terms of hydrogen-bonding interactions to the free N1, N2 and N3 backbone NH groups. Notably, the "good N2" amino acid residues Gln, Glu, Asp, Asn, Ser, Thr and His preferentially form i, i or i,i+1 hydrogen bonds to the backbone, though this is hindered by good N-caps (Asp, Asn, Ser, Thr and Cys) that compete for these hydrogen bond donors. We find a number of specific side-chain to side-chain interactions between N1 and N2 or between the N-cap and N2 or N3, such as Arg(N-cap) to Asp(N2). The strong energetic and structural preferences found for N1, N2 and N3, which differ greatly from positions within helix interiors, suggest that these sites should be treated explicitly in any consideration of helical structure in peptides or proteins.     

Engineering new peptidic inhibitors from a natural chymotrypsin inhibitor.

Three model peptides of different sizes (17-24 amino acid residues) mimicking the chymotrypsin inhibitor SCGI (a peptide of 35 amino acid residues) isolated from Schistocerca gregaria were designed and prepared by convergent peptide synthesis. Selective formation of disulphide bridges in the closing step was achieved without selective protection of cysteine residues. The natural pattern of the two disulphide bridges was determined by 2D homonuclear 1H NMR techniques. All three model peptides were characterized by amino acid analysis. MS and CD spectra. Preliminary results revealed that the two smaller model peptides exhibit no Inhibitory activity, whereas the larger one shows limited inhibition of chymotrypsin.     

Conformations of yeast alpha-mating factor and analog peptides as bound to phospholipid bilayer. Correlation of membrane-bound conformation with physiological activity

The transferred nuclear Overhauser effects of yeast alpha-mating factor [(1-13)peptide] in the presence of various spin-labeled phosphatidylcholines in small unilamellar vesicles of perdeuterated phosphatidylcholine have been analyzed. From the analysis of the quenching effect by spin-labels, the depth of amino acid side chains of the mating factor in phospholipid bilayer has been elucidated. The Leu4 and Leu6 residues are buried deeply in the apolar region of the phospholipid bilayer while the hydrophilic residues such as Gln5 and Lys7 are in the shallow region of the bilayer. The interaction of the side chains of Trp1 and Trp3 residues of alpha-mating factor with the hydrophobic interior of the bilayer contributes to the binding of this peptide with the phosphatidylcholine bilayer. The conformation of des-Trp1-alpha-mating-factor [(2-13)peptide] in the membrane-bound state has been found to be similar to that of (1-13)peptide from the analysis of transferred nuclear Overhauser effects in the presence of mixed vesicles of perdeuterated phosphatidylcholine and perdeuterated phosphatidylserine. The incorporation of this acidic phospholipid in the vesicle remarkably enhances the binding of (1-13)peptide and analog peptides. However, such modifications that weaken the interaction with phospholipid bilayer (deletion of Trp1 and substitution of Trp3 by Gly or Ala) appreciably lower the physiological activity. Transferred nuclear Overhauser effect analyses have also been made of [DHis2]peptide, [DLeu6]peptide and [DLys7]peptide in the presence of the vesicles of perdeuterated phosphatidylcholine. The main-chain conformations of these three analogs in the membrane-bound state have been found to be similar to that of (1-13)peptide, although the side-chain conformations of the D-amino acid residues are naturally different from those of the L-amino acid ones. Thus, the physiological activities of the (1-13)peptide and a variety of analog peptides are found to correlate with the affinities to the phosphatidylcholine/phosphatidylserine membrane and with the molecular conformations in the membrane-bound state.     

Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells.

To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (4(6), Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH( 2,) and Hel 11-7, KLLK-LLLK-LWKK-LLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 4(6) had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 4(6) and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide-DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.