Stereospecific binding of diastereomeric peptides to salmon sperm DNA.

Studies of the interaction specificities of L-lysyl-L-phenylalaninamide (1) and the diastereomeric dipeptide amide, L-lysyl-D-phenylalaninamide (2), with salmon sperm DNA reveal distinct differences in the binding site of the aromatic ring of the phenylalanine residue. The results of 1H nuclear magnetic resonance (NMR), spin-lattice relaxation rates, viscometric, and flow dichroism studies indicate the aromatic ring of 1 is "partially" inserted between base pairs of DNA whereas the aromatic ring of 2 points outward toward the solution. The terminal L-lysyl residue presumably interacts stereospecifically with DNA helix thus dictating the positioning of the aromatic ring of the C-terminal phenylalanine residue. In the accompanying paper (E. J. Gabbay et al. (1976), Biochemistry, following paper in this issue), the interaction of several oligopeptide amides (containing the N-terminal L-Lys-L-Phe residue) with DNA is examined. The results are found to be consistent with stereospecific binding of the terminal L-lysyl residue, and in addition, the evidence suggests that oligopeptides may bind to DNA via a modified single-stranded beta-sheet structure which is wrapped around the nucleic acid helix in a manner similar to that described by M. H. F. Wilkins (1956), Cold Spring Harbor Symp. Quant. Biol. 21, 75).     

The influence of intercalator structure on DNA binding strength: the importance of side chain orientation

A naphthothiophene intercalator with a cationic side chain linked to the ring through an ester group (1E) has been shown to bind to DNA almost an order of magnitude more strongly than a similar compound with the side chain linked to the ring through an amide group (1A) (W.D. Wilson, et al., Biophys. Chem. 24, 101-109 (1986]. X-ray crystallographic analysis of these two compounds indicates that both the ester and amide groups are essentially planar but that the amide is twisted approximately 30 degrees out of the aromatic plane of the naphthothiophene while the ester and ring system are co-planar. Proton NMR studies of the DNA complexes of these two compounds indicate that the naphthothiophene ring is intercalated in both 1A and 1E but that the protons of the ring system near the side chain interact with DNA base pairs at the binding site significantly better in 1E than in 1A. The protons next to the ester group on the side chain of 1E are also shifted upfield significantly more on addition of DNA than those of 1A. The large planar area of 1E, thus, allows greater stacking, complex geometry optimization, and dipolar interactions of the ester group with DNA base pairs at the binding site to account for the larger binding constant of this compound relative to 1A.     

Control of peptide conformation by the Thorpe-Ingold effect (C alpha-tetrasubstitution)

The preferred conformations of peptides heavily based on the currently extensively exploited achiral and chiral alpha-amino acids with a quaternary alpha-carbon atom, as determined by conformational energy computations, crystal-state (x-ray diffraction) analyses, and solution ((1)H-NMR and spectroscopic) investigations, are reviewed. It is concluded that 3(10)/alpha-helical structures and the fully extended (C(5)) conformation are preferentially adopted by peptide sequences characterized by this family of amino acids, depending upon overall bulkiness and nature (e.g., whether acyclic or C(alpha) (i) <--> C(alpha) (i) cyclized) of their side chains. The intriguing relationship between alpha-carbon chirality and bend/helix handedness is also illustrated. gamma-Bends and semiextended conformations are rarely observed. Formation of beta-sheet structures is prevented.     

Partial intercalation with DNA of peptides containing two aromatic amino acids

The interactions with DNA of tetrapeptide amides containing lysine at the N-terminal position and aromatic amino acids at the second and fourth positions (Ala at position three), 1-6, have been investigated by nmr, CD, and viscometric methods. Tetrapeptides with N-terminal lysine and a single aromatic amino acid, 7-10, were investigated as controls. Significant decreases in DNA viscosity occurred on addition of 7, with the aromatic group at the second position, but not with any of the other single aromatic amino acid peptides. All of the tetrapeptides with two aromatic groups caused DNA viscosity decreases which were two to three times larger than with 7. Peptides with p-nitrophenylalanine (p-NO2Phe) as the aromatic group were synthesized for nmr studies because of its simpler aromatic nmr spectrum relative to Phe. Large upfield shifts of the aromatic proton signals were obtained when the amino acid in the second position was L-p-NO2Phe, and the fourth position contained either p-NO2Phe or Phe. Such peptides also caused the largest DNA viscosity decreases on complex formation. Smaller upfield shifts of the aromatic signals were obtained when the amino acid in the second position was L-Phe or a D isomer of Phe or p-NO2Phe. With all peptides, larger upfield nmr shifts were obtained with heat-denatured, recooled DNA than with native DNA under the same conditions. As with nmr, CD results are quite different for the peptides with L and D amino acids at the second position. All of the results can be interpreted in terms of a model in which lysine interacts stereospecifically with the backbone in a DNA double helix and the aromatic group at the second position stacks strongly with the base pairs when the amino acid is an L isomer. The aromatic group at the fourth position can also interact with the base pairs, but primarily through a sideways stacking of the aromatic group with base pairs for either L or D isomers. Because of covalent constraints on the separation distance for the two aromatic groups in the tetrapeptides, they must stack on opposite sides of the same base pair in violation of the neighbor exclusion principle observed with classical intercalators. This stacking at the same base pair no doubt accounts for the larger viscosity decreases in DNA with the peptides containing two aromatic groups relative to those with a single aromatic group.(ABSTRACT TRUNCATED AT 400 WORDS)     

(alphaMe)Aun: a highly lipophilic, chiral, Calpha-tetrasubstituted alpha-amino acid. Incorporation into model peptides and preferred conformation.

Using a chemo-enzymatic approach we prepared the highly lipophilic, chiral, Calpha-methylated alpha-amino acid (alphaMe)Aun. Two series of terminally protected model peptides containing either D-(alphaMe)Aun in combination with Aib or L-(alphaMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT-IR absorption, 1H NMR and CD techniques, allowed us to determine the preferred conformation of this amino acid and the relationship between chirality at its alpha-carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that D-(alphaMe)Aun favors the formation of the left-handed 3(10)-helical conformation.     

Novel cyclization chemistry especially suited for biologically derived, unprotected peptides.

A novel method is described for the cyclization of peptides--or segments of polypeptides--which requires a free N-terminal alpha-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal alpha-amino group with iodoacetic anhydride at pH 6. This acylation reaction has greater than 90% specificity for peptide alpha-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al., Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular architecture of the undecameric rotor of a bacterial Na+-ATP synthase

The sodium ion-translocating F(1)F(0) ATP synthase from the bacterium Ilyobacter tartaricus contains a remarkably stable rotor ring composed of 11 c subunits. The rotor ring was isolated, crystallised in two dimensions and analysed by electron cryo-microscopy. Here, we present an alpha-carbon model of the c-subunit ring. Each monomeric c subunit of 89 amino acid residues folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop. The 11 N-terminal helices are closely spaced within an inner ring surrounding a cavity of approximately 17A (1.7 nm). The tight helix packing leaves no space for side-chains and is accounted for by a highly conserved motif of four glycine residues in the inner, N-terminal helix. Each inner helix is connected by a clearly visible loop to an outer C-terminal helix. The outer helix has a kink near the position of the ion-binding site residue Glu65 in the centre of the membrane and another kink near the C terminus. Two helices from the outer ring and one from the inner ring form the ion-binding site in the middle of the membrane and a potential access channel from the binding site to the cytoplasmic surface. Three possible inter-subunit ion-bridges are likely to account for the remarkable temperature stability of I.tartaricus c-rings compared to those of other organisms.     

Preferred solution conformation of peptides rich in the lipophilic, chiral, C(alpha)-methylated alpha-amino acid (alpha Me)Aoc.

The lipophilic, chiral, C(alpha)-methylated alpha-amino acid L-(alphaMe)Aoc (2-methyl-2-amino-octanoic acid) was prepared using a chemo-enzymatic approach. Two series of terminally protected model peptides, from dimer through to hexamer, containing L-(alphaMe)Aoc in combination with either Gly or Aib, were synthesized by solution methods and were fully characterized. A solution conformational analysis, based on FT-IR absorption, 1H-NMR and circular dichroism (CD) techniques, was performed with the aim at determining the preferred conformation of this novel amino acid and the relationship between chirality at its alpha-carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that L-(alphaMe)Aoc favours the formation of the right-handed 3(10)-helical conformation.     

Construction and design of single stranded collagen-like structure

Polytheonamide B, a 48 residue long highly cytotoxic polypeptide extracted from marine sponges contains amino acids of alternate chirality and the N-terminal region is rich in t-Leu residues. The aim of this study is to analyze the effect of these alternate chiralities and conformational behavior of various model peptides containing t-Leu, in order to explore their role in designing bioactive peptides that shall offer advantages comparable to polytheonamide B, while circumventing its limitations. The conformational behavior of various peptides constructed from t-Leu of the form Ac-(L/D-X-L/D-Y)n-NHMe, where X = Gly/Ala/Leu and Y = t-Leu has been studied and compared with the corresponding peptides containing Leu residue. The results show that the helix driving capacity of L and D forms of t-Leu is less than that of Leu residue. In poly t-Leu peptides, the population of collagen/inverse collagen-type structures or right/left handed-helical structures for L and D forms respectively is found to be chain length-dependent. The stability of the helical structures is increased by -2 kcal per residue over the collagen-type structure in poly t-Leu peptides with chain length greater than five residues. Molecular view of peptides in collagen-type structure shows that the bulky side chains of t-Leu residues mask the NH moieties of the peptide bond, while the carbonyl groups lying along the helical groove are accessible to the small solvent molecules. Molecular model building suggests that one ethylene glycol molecule interacts by forming hydrogen bonds with carbonyl groups of two adjacent t-Leu residues. To the best of our knowledge, this is the first study of its own kind on the construction of a single-strand collagen/inverse collagen-type structure using unusual amino acid residues. Such synthetic collagen mimetic peptides shall exhibit specific affinity to natural collagen under controlled thermal conditions (heat or laser treatment) and hence can be explored as a new targeting method to attach therapeutic drugs to collagens in the living tissues and to biomaterials that incorporate natural collagens.     

Enkephalin analogues containing beta-naphthylalanine at the fourth position

To examine the importance of the aromatic side chains of enkephalin on opiate activity, we report the synthesis and conformational analysis of a series of analogues related to enkephalin with beta-naphthylalanine in place of phenylalanine at the fourth position. Three linear analogues (Tyr-D-Ala-Gly-(L and D)-beta Nal(1)-Leu-NH2 and Tyr-D-Ala-Gly-beta Nal(2)-Leu-NH2) were initially synthesized to examine the effect of the substitution on biological activity. The increased activity of these peptides at the mu-opiate receptor, compared to native Leu-enkephalin, prompted us to examine the more conformational constrained analogues, Tyr-c[D-A2bu-Gly-(L and D)-beta Nal(1)-Leu], incorporating a alpha, gamma-diaminobutyric acid at the second position and cyclization to the carboxylic end of the leucine. These two cyclic analogues provide insight into the necessity for the L chirality of the aromatic residue at position 4. The Tyr-c[D-A2bu-Gly-L-beta Nal(1)-Leu] analogue is highly potent and displays a slight preference for the mu receptor. The conformational analysis indicates that despite the high flexibility of the tyrosine side chain, the aromatic rings of the tyrosine and naphthylalanine are relatively distant from each other. The presence of two intramolecular hydrogen bonds help maintain the conformation of the 14-membered backbone ring that keeps the side chains directed away from each other. These findings are in agreement with our model of an extended structure required for mu selectivity and a folded form with close aromatic ring placement for delta selectivity.     

Hairpin and duplex forms of a self-complementary dodecamer, d-AGATCTAGATCT, and interaction of the duplex form with the peptide KGWGK: can a pentapeptide destabilize DNA?

Ordered forms of a synthetic dodecamer, d-AGATCTAGATCT, a direct repeat of the BglII recognition sequence, have been investigated using UV, CD, and fluorescence spectroscopy. Complex hairpin-duplex equilibria are manifest in UV thermal transitions, which are monophasic in the presence of very low or high NaCl concentrations but distinctly biphasic at intermediate ionic strengths. In 100 mM NaCl, the 1/Tm vs 1n C curve has a reasonable positive slope, which yields delta H and delta S for duplex formation as -66.2 kcal/mol and -190 cal/mol, respectively. Interaction of the dodecamer in duplex form with a tryptophan-containing peptide, KGWGK, has also been investigated to test the "bookmark" hypothesis (Gabbay et al., 1976) under the uniform structural constraint of the oligonucleotide of defined sequence. CD spectra of the peptide (P), the oligonucleotide (N), and their mixtures at different P/N ratios show a dramatic change in peptide spectrum but little change in nucleic acid dichroism with peptide binding. The Tm of P-N complexes decreases with an increase in peptide binding and levels off at saturation binding of P/N = 2.0. The data are interpreted in terms of a groove-cum-intercalation mode of binding, where intercalation to the tryptophan side chain destabilizes the double helix. A Scatchard plot of the binding data is nonlinear, with best-fit values for an overall association constant K = 4.33 x 10(5) M-1, and the number of binding sites n = 3.23 when fitted to the site-exclusion model of binding.     

Influence of chirality,amino acid sequence,and protecting groups on the intramolecular interaction behavior of dipeptide and tripeptide derivatives

The intramolecular interaction of protected dipeptides and tripeptides containing the amino acid units Ala, Phe, and Val was studied by means of ir spectroscopy. The NH and CO regions of the compounds dissolved in carbon tetrachloride clearly show the existence of different intramolecular hydrogen bonds. Using solvents with higher polarity such as chloroform and methylene chloride, the association bands disappear. Investigating the substances with the same amino acid sequence but opposite chirality of the central C atom in the peptide chain, we observed different band shapes in the CO and NH regions. Large effects were found when the chirality of the Phe unit in the second position was changed. This is probably due to the steric hindrance originated by the rotation of the aromatic ring in the side chain. The protecting groups, Z (benzyloxycarbonyl) or Boc (tert-butyloxycarbonyl) residues at the N-terminal group and methyl- or tert-butyl esters at the C-terminal group, influence the solubility of the substances in nonpolar solvent, as well as the NH and CO association band profiles in the methylene chloride solutions. The consequences of changing the sequence of the amino acids are discussed for the tripeptide derivatives. Besides a qualitative discussion, some quantitative considerations concerning the intramolecular interaction are also given to illustrate the different stabilities of the associates. © 1996 John Wiley & Sons, Inc.     

Influence of hydrogen bonding,main chain–side chain interactions,and protecting groups on the excimer formation of bis(pyrenylalanine) peptides

Dipeptides of the aromatic fluorescent amino acid, pyrenylalanine, are studied using both stationary and transient fluorescence techniques. Since the conformational transitions of the peptide chain are slow compared to the decay of the pyrene excited state, both ground state conformations, adopted by the peptide, i.e., C₅ and C₇, can be monitored separately. Kinetic models are proposed to describe the molecular dynamics of the peptide chain as probed by the intramolecular excimer formation between both pyrene chromophores. These kinetic schemes explain the influence of solvent, chain chirality, main chain–side chain interactions, and nature of the protecting groups on the emission spectrum and the fluorescence decay profile of these model peptides. These schemes also provide a tool to calculate rate constants of conformational transitions and excimer formation. By comparing the kinetic and thermodynamic parameters of the various compounds, the influence of a structural modification on the molecular dynamics of the peptide chain is determined.     

Noncovalent chiral domino effect on one-handed helix of nonapeptide containing a midpoint L-residue

We here clarify whether noncovalent chiral domino effect characterized by the terminal interaction of a helical peptide with a chiral small molecule can alter the helical stability of N-deprotected peptides containing an L-residue covalently incorporated into the inner position. Two nonapeptides consisting of the midpoint L-leucine (1) or L-phenylalanine (2) and the achiral helix-forming residues were employed. NMR and IR spectroscopy and energy calculation indicated that both peptides adopt a 3(10)-helical conformation in chloroform. They strongly preferred a right-handed screw sense because of the presence of the midpoint L-residue. These original right-handed screw senses were retained on addition of chiral Boc-amino acid, but their helical stabilities clearly depended on its added chirality. Here, Boc-L-amino acid stabilizes the original right-handed helix, whereas the corresponding Boc-D-amino acid tends to less stabilize or destabilize it. This tendency was not observed for the corresponding N-Boc-protected peptides 1 and 2, strongly suggesting that the N-terminal amino group is required for controlling the stabilization of the original right-handed helix. Therefore, noncovalent chiral domino effect in peptides 1 and 2 can contribute even to the helical stability of a chiral peptide prevailing one-handed helix strongly through the midpoint L-residue. In addition, the N-terminal moiety of a 3(10)-helical peptide was found to generate chiral discrimination in complexation process with racemic additives.     

Chiral interaction in peptide molecules: effects of chiral peptide species on helix-sense induction in an N-terminal-free achiral peptide

Noncovalent chiral domino effect (NCDE) has been proposed as terminal-specific interaction upon a 3(10)-helical peptide chain, of which the helix sense is manipulated by an external chiral stimulus (mainly amino acid derivatives) operating on the N-terminus (Inai, Y., et al. J Am Chem Soc 2000, 122, 11731-11732; ibid., 2002, 124, 2466-2473; ibid., 2003, 125, 8151-8162). We have investigated here a helix-sense induction in an optically inactive N-terminal-free nonapeptide (1) through the screening of several peptide species that differ in chiral sequence, chain length, and C-terminal group. Helix-sense induction in peptide 1 depends largely on both the C-terminal chirality and carboxyl group in the external peptide, in which N-carbonyl-blocked amino acids, "monopeptide acids," should be the minimum requirement for effective induction. N-Protected mono- to tetrapeptides of L-Leu residue commonly induce a right-handed helix. NMR study and theoretical computation reveal that the N-terminal segment of peptide 1 binds the N-protected dipeptide molecule through multipoint coordination to induce a right-handed helix preferentially. The present findings not only will improve our understanding of the chiral roles in peptide or protein helical termini, but also might demonstrate potential applications to chirality-responsive materials based on peptide helical fragments.     

Solid-phase synthesis of an Htc-containingdimer analog of the autophosphorylationsite of pp60src PTK: Effective acylationconditions for Htc residues

Summary The difficulty during SPPS in acylating the secondary amino group of Htc, a locally constrained tyrosine, can be correlated with the steric hindrance of the amino acid or with the conformation of the growing peptide chain. Our experimental data indicate that the availability of the Htc amino group is associated with its steric hindrance rather than a conformational effect of the peptide chain. An optimized solid phase automated protocol for Htc is reported. Under optimal conditions, Fmoc-amino acids with hindered side chains were incorporated in approximately 99% yield using HATU as coupling reagent. Unhindered side chain amino acid acylated the secondary amino group of Htc in good yield under classical HBTU/HOBt coupling conditions. Abbreviations: Abbreviations used for amino acids and the designation of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977–983]. Amino acid symbols denote thel-configuration where applicable, unless indicated otherwise. The following additional, abbreviations are used     

Helix propensities of the amino acids measured in alanine-based peptides without helix-stabilizing side-chain interactions.

Helix propensities of the amino acids have been measured in alanine-based peptides in the absence of helix-stabilizing side-chain interactions. Fifty-eight peptides have been studied. A modified form of the Lifson-Roig theory for the helix-coil transition, which includes helix capping (Doig AJ, Chakrabartty A, Klingler TM, Baldwin RL, 1994, Biochemistry 33:3396-3403), was used to analyze the results. Substitutions were made at various positions of homologous helical peptides. Helix-capping interactions were found to contribute to helix stability, even when the substitution site was not at the end of the peptide. Analysis of our data with the original Lifson-Roig theory, which neglects capping effects, does not produce as good a fit to the experimental data as does analysis with the modified Lifson-Roig theory. At 0 degrees C, Ala is a strong helix former, Leu and Arg are helix-indifferent, and all other amino acids are helix breakers of varying severity. Because Ala has a small side chain that cannot interact significantly with other side chains, helix formation by Ala is stabilized predominantly by the backbone ("peptide H-bonds"). The implication for protein folding is that formation of peptide H-bonds can largely offset the unfavorable entropy change caused by fixing the peptide backbone. The helix propensities of most amino acids oppose folding; consequently, the majority of isolated helices derived from proteins are unstable, unless specific side-chain interactions stabilize them.     

External chirality-triggered helicity control promoted by introducing a beta-Ala residue into the N-terminus of chiral peptides

The noncovalent chiral domino effect (NCDE), defined as chiral interaction upon an N-terminus of a 3(10)-helical peptide, will provide a unique method for structural control of a peptide helix through the use of external chirality. On the other hand, the NCDE has not been considered to be effective for the helicity control of peptides strongly favoring a one-handed screw sense. We here aim to promote the NCDE on peptide helicity using two types of nonapeptides: H-beta-Ala-Delta(Z)Phe-Aib-Delta(Z)Phe-X-(Delta(Z)Phe-Aib)(2)-OCH(3) [Delta(Z)Phe = alpha,beta-didehydrophenylalanine, Aib = alpha-aminoisobutyric acid], where X as the single chirality is L-leucine (1) or L-phenylalanine (2). NMR, IR, and CD spectroscopy as well as energy calculation revealed that both peptides alone form a right-handed 3(10)-helix. The original CD amplitudes or signs in chloroform, irrespective of a strong screw-sense preference in the central chirality, responded sensitively to external chiral information. Namely added Boc-L-amino acid stabilized the original right-handed helix, while the corresponding d-isomer destabilized it or transformed it into a left-handed helix. These peptides were also shown to bind more favorably to an L-isomer from the racemate. Although similar helicity control was observed for analogous nonapeptides bearing an N-terminal Aib residue (Inai, Y.; et al. Biomacromolecules 2003, 4, 122), the present findings demonstrate that the N-terminal replacement by the beta-Ala residue significantly improves the previous NCDE to achieve more effective control of helicity. Semiempirical molecular orbital calculations on complexation of peptide 2 with Boc-(L or D)-Pro-OH reasonably explained the unique conformational change induced by external chirality.     

Sequence-dependent binding of bis-amidine carbazole dications to DNA.

The conventional wisdom argues that DNA intercalators possess a condensed polyaromatic ring whereas DNA minor groove binders generally contain unfused aromatic heterocycles, frequently separated by amide bonds. Recently, this view has been challenged with the discovery of powerful intercalating agents formed by unfused aromatic molecules and groove binders containing a polyaromatic nucleus. Bis-amidinocarbazoles belong to this later category of drugs having a planar chromophore and capable of reading the genetic information accessible within the minor groove of AT-rich sequences [Tanious, F.A., Ding, D., Patrick, D.A., Bailly, C., Tidwell, R.R. & Wilson, W.D. (2000) Biochemistry 39, 12091-12101]. But in addition to the tight binding to AT sites, we show here that bis-amidinocarbazoles can also interact with GC sites. The extent and mode of binding of 2,7 and 3,6 substituted amidinocarbazoles to AT and GC sequences were investigated by complementary biochemical and biophysical methods. Absorption, fluorescence, melting temperature and surface plasmon resonance (SPR) measurements indicate that the position of the two amidine groups on the carbazole ring influences significantly the drug-DNA interaction. SPR and DNase I footprinting data confirm the AT-preference of the compounds and provide useful information on their additional interaction with GC sequences. The 3,6-carbazole binds approximately twice as strongly to the GC-containing hairpin oligomer than the 2,7-regioisomer. The high tendency of the 3,6 compound to intercalate into different types of DNA containing G.C base pairs is shown by electric linear dichroism. This work completes our understanding of the sequence-dependent DNA binding properties of carbazole dications.     

Bacterial beta-peptidyl aminopeptidases with unique substrate specificities for beta-oligopeptides and mixed beta,alpha-oligopeptides

We previously discovered that BapA, a bacterial beta-peptidyl aminopeptidase, is able to hydrolyze two otherwise metabolically inert beta-peptides [Geueke B, Namoto K, Seebach D and Kohler H-PE (2005) J Bacteriol 187, 5910-5917]. Here, we describe the purification and characterization of two distinct bacterial beta-peptidyl aminopeptidases that originated from different environmental isolates. Both bapA genes encode a preprotein with a signal sequence and were flanked by ORFs that code for enzymes with similar predicted functions. To form the active enzymes, which had an (alphabeta)(4) quaternary structure, the preproteins needed to be cleaved into two subunits. The two beta-peptidyl aminopeptidases had 86% amino acid sequence identity, hydrolyzed a variety of beta-peptides and mixed beta/alpha-peptides, and exhibited unique substrate specificities. The prerequisite for peptides being accepted as substrates was the presence of a beta-amino acid at the N-terminus; peptide substrates with an N-terminal alpha-amino acid were not hydrolyzed at all. Both enzymes cleaved the peptide bond between the N-terminal beta-amino acid and the amino acid at the second position of tripeptidic substrates of the general structure H-betahXaa-Ile-betahTyr-OH according to the following preferences with regard to the side chain of the N-terminal beta-amino acid: aliphatic and aromatic > OH-containing > hydrogen, basic and polar. Experiments with the tripeptides H-d-betahVal-Ile-betahTyr-OH and H-betahVal-Ile-betahTyr-OH demonstrated that the two BapA enzymes preferred the peptide with the l-configuration of the N-terminal beta-homovaline residue as a substrate.