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The structural basis for the gram selectivity of two disulfide-bonded β-hairpin antimicrobial peptides (AMPs) is investigated using solid-state nuclear magnetic resonance (NMR) spectroscopy. The hexa-arginine PG-1 exhibits potent activities against both gram-positive and gram-negative bacteria, while a mutant of PG-1 with only three cationic residues maintains gram-positive activity but is 30-fold less active against gram-negative bacteria. We determined the topological structure and lipid interactions of these two peptides in a lipopolysaccharide (LPS)-rich membrane that mimics the outer membrane of gram-negative bacteria and in the POPE/POPG membrane, which mimics the membrane of gram-positive bacteria. (31)P NMR line shapes indicate that both peptides cause less orientational disorder in the LPS-rich membrane than in the POPE/POPG membrane. (13)C chemical shifts and (13)C-(1)H dipolar couplings show that both peptides maintain their β-hairpin conformation in these membranes and are largely immobilized, but the mutant exhibits noticeable intermediate-time scale motion in the LPS membrane at physiological temperature, suggesting shallow insertion. Indeed, (1)H spin diffusion from lipid chains to the peptides shows that PG-1 fully inserts into the LPS-rich membrane whereas the mutant does not. The (13)C-(31)P distances between the most hydrophobically embedded Arg of PG-1 and the lipid (31)P are significantly longer in the LPS membrane than in the POPE/POPG membrane, indicating that PG-1 does not cause toroidal pore defects in the LPS membrane, in contrast to its behavior in the POPE/POPG membrane. Taken together, these data indicate that PG-1 causes transmembrane pores of the barrel-stave type in the LPS membrane, thus allowing further translocation of the peptide into the inner membrane of gram-negative bacteria to kill the cells. In comparison, the less cationic mutant cannot fully cross the LPS membrane because of weaker electrostatic attractions, thus causing weaker antimicrobial activities. Therefore, strong electrostatic attraction between the peptide and the membrane surface, ensured by having a sufficient number of Arg residues, is essential for potent antimicrobial activities against gram-negative bacteria. The data provide a rational basis for controlling gram selectivity of AMPs by adjusting the charge densities.

Interleukin-8alpha (IL-8alpha) is an antimicrobial peptide derived from the chemokine IL-8. Solution NMR was used to determine the atomic-resolution structure of IL-8alpha in SDS micelles. Solid-state NMR and tryptophan fluorescence were used to probe the interaction of IL-8alpha with model membranes. The peptide interacted differently with anionic versus purely zwitterionic micelles or bilayers. Tryptophan fluorescence demonstrated a deeper position of Trp4 in SDS micelles and POPC/POPG bilayers compared to pure POPC bilayers, consistent with (2)H order parameters, which also indicated a deeper position of the peptide in POPC/POPG bilayers compared to POPC bilayers. Paramagnetic probe data showed that IL-8alpha was situated roughly parallel to the SDS micelle surface, with a slight tilt that positioned the N-terminus more deeply in the micelle compared to the C-terminus. (15)N solid-state NMR spectra indicated a similar, nearly parallel position for the peptide in POPC/POPG bilayers. (31)P and (2)H solid-state NMR demonstrated that the peptide did not induce the formation of any nonlamellar phases and did not significantly disrupt bilayer orientation in aligned model membranes composed of POPC or POPC and POPG.

Cell-penetrating peptides (CPPs) are small cationic peptides that cross the cell membrane while carrying macromolecular cargoes. We use solid-state NMR to investigate the structure and lipid interaction of two cationic residues, Arg(10) and Lys(13), in the CPP penetratin. (13)C chemical shifts indicate that Arg(10) adopts a rigid beta-strand conformation in the liquid-crystalline state of anionic lipid membranes. This behavior contrasts with all other residues observed so far in this peptide, which adopt a dynamic beta-turn conformation with coil-like chemical shifts at physiological temperature. Low-temperature (13)C-(31)P distances between the peptide and the lipid phosphates indicate that both the Arg(10) guanidinium Czeta atom and the Lys(13) Cepsilon atom are close to the lipid (31)P (4.0-4.2 A), proving the existence of charge-charge interaction for both Arg(10) and Lys(13) in the gel-phase membrane. However, since lysine substitution in CPPs is known to weaken their translocation ability, we propose that the low temperature stabilizes interactions of both lysine and arginine with the phosphates, whereas at high temperatures, the lysine-phosphate interaction is much weaker than the arginine-phosphate interaction. This is supported by the unusually high rigidity of the Arg(10) side chain and its beta-strand conformation at high temperatures. The latter is proposed to be important for ion pair formation by allowing close approach of the lipid headgroups to guanidinium side chains. (19)F and (13)C spin diffusion experiments indicate that penetratin is oligomerized into beta-sheets in gel-phase membranes. These solid-state NMR data indicate that guanidinium-phosphate interactions exist in penetratin, and guanidinium groups play a stronger structural role than ammonium groups in the lipid-assisted translocation of CPPs across liquid-crystalline cell membranes.

Surfactant protein B (SP-B) is essential for normal lung surfactant function, which is in itself essential to life. However, the molecular basis for SP-B's activity is not understood and a high-resolution structure for SP-B has not been determined. Mini-B is a 34-residue peptide with internal disulfide linkages that is composed of the N- and C-terminal helical regions of SP-B. It has been shown to retain similar activity to full-length SP-B in certain in vitro and in vivo studies. We have used solution NMR to determine the structure of Mini-B in the presence of micelles composed of the anionic detergent sodium dodecyl sulfate (SDS). Under these conditions, Mini-B forms two alpha-helices connected by an unstructured loop. Mini-B possesses a strikingly amphipathic surface with a large positively charged patch on one face of the peptide and a large hydrophobic patch on the opposite face. A tryptophan side chain extends outward from the peptide in a position to interact with lipids at the polar/apolar interface. Interhelix interactions are stabilized by both disulfide bonds and by interleaving of hydrophobic side chains from the two helices.

The interaction of a beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), with various lipid membranes is investigated by (31)P, (2)H, and (13)C solid-state NMR. Mixed lipid bilayers containing anionic lipids and cholesterol are used to mimic the bacterial and mammalian cell membranes, respectively. (31)P and (2)H spectra of macroscopically oriented samples show that PG-1 induces the formation of an isotropic phase in anionic bilayers containing phosphatidylglycerol. Two-dimensional (31)P exchange experiments indicate that these isotropic lipids are significantly separate from the residual oriented lamellar bilayers, ruling out toroidal pores as the cause for the isotropic signal. (1)H spin diffusion experiments show that PG-1 is not exclusively bound to the isotropic phase but is also present in the residual oriented lamellar bilayers. This dynamic and morphological heterogeneity of the anionic membranes induced by PG-1 is supported by the fact that (13)C T(2) relaxation times measured under cross polarization and direct polarization conditions differ significantly. In contrast to the anionic membrane, the zwitterionic phosphatidylcholine (PC) membrane does not form an isotropic phase in the presence of PG-1 but shows significant orientational disorder. The addition of cholesterol to the PC bilayer significantly reduces this orientational disorder. The (13)C T(2) relaxation times of the PC lipids in the presence of both cholesterol and PG-1 suggest that the peptide may decrease the dynamic heterogeneity of the cholesterol-containing membrane. The observed selective interaction of PG-1 with different lipid membranes is consistent with its biological function and may be caused by its strong cationic and amphipathic structure.

We study the interaction of antimicrobial peptides with lipopolysaccharide (LPS) bilayers to understand how antimicrobial peptides interact with the LPS monolayer on the outer membrane of Gram-negative bacteria. LPS in water spontaneously forms a multilamellar structure composed of symmetric bilayers. We performed X-ray lamellar diffraction and wide-angle in-plane scattering to study the physical characteristics of LPS multilayers. The multilayer alignment of LPS is comparable to phospholipids. Thus, it is suitable for the application of oriented circular dichroism (OCD) to study the state of peptides in LPS bilayers. At high hydration levels, the chain melting temperature in multilamella detected by X-ray diffraction is the same as that of LPS aqueous dispersions, as measured by calorimetry. LPS has a strong CD, but with a careful subtraction of the lipid background, the OCD of peptides in LPS is measurable. The method was tested successfully with melittin. It was then applied to two representative antimicrobial peptides, magainin and protegrin. At peptide concentrations comparable to the physiological conditions, both peptides penetrate transmembrane in LPS bilayers. The results imply that antimicrobial peptides readily penetrate the LPS monolayer of the outer membrane.

Protegrin-1 (PG-1) is a broad-spectrum beta-sheet antimicrobial peptide found in porcine leukocytes. The mechanism of action and the orientation of PG-1 in lipid bilayers are here investigated using (2)H, (31)P, (13)C, and (15)N solid-state NMR spectroscopy. (2)H spectra of mechanically aligned and chain-perdeuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers indicate that PG-1 at high concentrations destroys the orientational order of the aligned lamellar bilayer. The conformation of the lipid headgroups in the unoriented region is significantly altered, as seen from the (31)P spectra of POPC and the (2)H spectra of headgroup-deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine. These observations indicate that PG-1 disrupts microbial membranes by breaking the extended bilayer into smaller disks, where a significant fraction of lipids is located in the edges of the disks with a distribution of orientations. These edges allow the lipid bilayer to bend back on itself as in toroidal pores. Interestingly, this loss of bilayer orientation occurs only in long-chain lipids such as POPC and not in shorter chain lipids such as 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC). To understand the mode of binding of PG-1 to the lipid bilayer, we determined the orientation of PG-1 in DLPC bilayers. The (13)CO and (15)N chemical shifts of Val-16 labeled PG-1 indicate that the beta-strand axis is tilted by 55 degrees +/- 5 degrees from the bilayer normal while the normal of the beta-sheet plane is 48 degrees +/- 5 degrees from the bilayer normal. This orientation favors interaction of the hydrophobic backbone of the peptide with the hydrophobic core of the bilayer and positions the cationic Arg side chains to interact with the anionic phosphate groups. This is the first time that the orientation of a disulfide-stabilized beta-sheet membrane peptide has been determined by solid-state NMR.

Lipid bilayers containing the antimicrobial peptide protegrin-1 (PG-1) were studied by lamellar X-ray diffraction. Previously, we have shown that the peptide exists in two distinct states when associated with lipid bilayers depending on the peptide concentration [Heller, W. T., Waring, A. J., Lehrer, R. I., and Huang, H. W. (1998) Biochemistry 37, 17331-17338]. For concentrations below a lipid-dependent threshold, PG-1 exhibits a unique oriented circular dichroism spectrum called the S state. X-ray experiments show that in this state PG-1 decreases the thickness of the lipid bilayer in proportion to the peptide concentration, similar to alamethicin's membrane thinning effect. This indicates that the S state is adsorbed in the headgroup region of the lipid bilayer, where the peptide is in an inactive state. For PG-1 above the threshold concentration, X-ray diffraction shows that the interaction between the peptide and the bilayer changes significantly. These results suggest that PG-1 has the same concentration-gated mechanism of action as alamethicin.

Permeabilized preparations of equine infectious anemia virus (EIAV) are shown here to support efficient and accurate synthesis of full-length double-stranded proviral DNA. When (-) and (+) strand products were analyzed by Southern blotting, a discontinuity, mapping approximately to the center of the EIAV genome, could be demonstrated for the (+) strand, predicting a second site for initiation of DNA synthesis and a specific mechanism of (+) strand termination. Precise localization of this (+) strand origin within the integrase (IN) coding region was achieved through its in vitro selection and extension into, and excision from, nascent DNA by purified recombinant p66/p51 EIAV reverse transcriptase (RT), suggesting that the EIAV genome harbors a central polypurine tract (cPPT). In addition, a model system was developed for evaluating whether sequences immediately downstream of the cPPT would terminate (+) strand synthesis in the context of strand displacement. Such a sequence was indeed discovered which functions in a manner analogous to that of the central termination sequence (CTS) of HIV, where A-tract-induced minor groove compression has been suggested to induce localized distortion of the nucleic acid duplex and termination of (+) strand synthesis. This interpretation is reinforced by experiments indicating that read-through of the CTS can be efficiently promoted by substituting 2,6-diaminopurine for adenine, thereby relieving minor groove compression. The nucleotide substitution can also shift the site of termination in strand displacement (+) strand synthesis. Collectively, our data support proposals that lentiviruses may have evolved specialized mechanisms for initiating and terminating (+) strand DNA synthesis at the center of their genomes.

Oligonucleotide-directed triple helix formation normally requires a long tract of oligopyrimidine.oligopurine sequence. This limitation can be partially overcome by alternate-strand triple helix (or switch triple helix) formation which enables recognition of alternating oligopurine/oligopyrimidine sequences. The present work is devoted to the optimization of switch triple helix formation at the 5'CpG3' and 5'GpC3' junction steps by combination of base triplets in Hoogsteen and in reverse Hoogsteen configurations. Rational design by molecular mechanics was first carried out to study the geometrical constraints at different junction steps and to propose a "switch code" which would optimize the interactions at junctions. These predictions were further checked and validated experimentally by gel retardation and DNase I footprinting assays. It was shown that the choice of an appropriate linker nucleotide in the switching third strand plays an important role in the interaction between oligonucleotides and alternating oligopurine/oligopyrimidine target sequences at different junctions: (i) the addition of a cytosine at the junction level in the oligonucleotide optimizes the crossover at the 5'CpG3' junction, whereas (ii) the best crossover at the 5'GpC3' junction step is achieved without any additional nucleotide. These results provide a useful guideline to extend double-stranded DNA sequence recognition by switch triple helix formation.

The binding of the anticancer drug daunomycin to double-helical DNA has been investigated by DNase I footprinting and fluorescence titration, using a series of polymerase chain reaction (PCR) synthesized DNA fragments that contained systematic base substitutions to alter the disposition of functional groups within the minor groove. The 160 bp tyrT DNA fragment constituted the starting material. Fragments in which (i) inosine was substituted for guanosine, (ii) diaminopurine was substituted for adenine, and (iii) both inosine and diaminopurine were substituted for guanosine and adenine, respectively, were studied. These fragments permit the role of the 2-amino group in the minor groove to be systematically explored. The results of DNase I footprinting experiments confirmed that daunomycin binds preferentially to 5'(A/T)GC and 5'(A/T)CG triplets in the normal fragment. Substitution of inosine for guanosine, with the concomitant loss of the N-2 in the minor groove, weakened binding affinity but did not dramatically alter the sequence preference associated with daunomycin binding. Complete reversal of the location of the N-2 group by the double substitution, however, completely altered the sequence preference of daunomycin and shifted its binding from the canonical triplets to ones with a 5'IDD motif. These results have critically tested and confirmed the proposed key roles of the daunosamine moiety and the 9-OH group of daunomycin in dictating binding to preferred sites. In a parallel study, both macroscopic and microscopic binding to the normal tyrT fragment were investigated, experiments made possible by using PCR to prepare large quantities of the long, defined DNA sequence. The results of these experiments underscored the complexity of the interaction of the drug with the DNA lattice and revealed unequivocal heterogeneity in its affinity for different binding sites. A class of high-affinity sites, most probably corresponding to the 5'(A/T)GC and 5'(A/T)CG triplets, was identified and characterized in macroscopic binding isotherms.

Combilexins are a group of DNA ligands having a sequence-specific minor groove binding element combined with an intercalating chromophore which stabilizes the DNA complex and can interfere with topoisomerases. In this study, complementary methods of spectroscopy (absorption, circular dichroism, electric linear dichroism) and biochemistry (viscometry, footprinting) have been applied to explore the nature of the complex formed between a new amsacrine-4-carboxamide-netropsin combilexin and DNA or chromatin. Collectively, the structural and kinetic data concur that the conjugate threads through the DNA double helix so as to intercalate its acridine chromophore, leaving the netropsin moiety and the methanesulfonanilino group positioned within the minor and major grooves of the double helix, respectively. The hybrid retains the AT selectivity conferred by the netropsin moiety. The threading-type intercalation process, evidenced by stopped-flow measurements, is affected when the DNA is wrapped around histones. The composite drug can bind to both the DNA linker segments and the nucleosomal cores in chromatin though, unlike its constituents, it antagonizes the salt-induced condensation of chromatin. As far as its mode of binding to DNA is concerned, the netropsin-amsacrine hybrid molecule exhibits structural features reminiscent of the antitumor antibiotics nogalamycin and pluramycin. The design of DNA-threading combilexins provides an original route for the development of sequence-specific ligands capable of forming stable complexes with DNA.

Quantitative footprinting has been used to investigate cooperative binding of the antitumor antibiotic echinomycin to DNA fragments containing closely spaced CpG steps. The sequences of the designed DNA fragments contained two pairs of strong echinomycin binding sites: a pair of ACGT sites together with an ACGT site and a TCGA site, either directly adjacent or separated by two or four A.T base pairs. The results demonstrate that the binding of echinomycin to the sequences ACGTACGT and TCGAACGT is highly cooperative. The extent of cooperativity depends on the nature of the sequences clamped by the antibiotic and diminishes as the distance between the binding sites is increased. Various methods of extracting the information necessary to establish cooperativity have been compared. Beyond the specific interest in echinomycin-DNA interaction, the present quantitative footprinting study provides a model that may be generally applicable for designing investigations into cooperativity in drug-DNA recognition.

The DNA binding properties and effects on topoisomerase II of MePyGA, an anilinoacridine derivative bearing an N-methylpyrrolecarboxamide unit at position 1', have been compared with those of its precursor glycylanilinoacridine and the structurally related antileukaemic drug amsacrine. Electric linear dichroism spectroscopy reveals that MePyGA intercalates its acridine chromophore between DNA base pairs with a preference for GC-rich sequences, whereas both its structural analogue lacking the N-methylpyrrole unit and amsacrine intercalate into DNA without any strong sequence preference. The effects of the test drug on the catalytic activities of topoisomerase II were studied in vitro using purified calf thymus enzyme and 32P-labeled DNA. MePyGA stabilizes the topoisomerase II-DNA covalent complex and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. The removal of the N-methylpyrrole unit abolishes both the GC-preferential binding to DNA and the topoisomerase II-mediated DNA cleavage. MePyGA and amsacrine stimulate the cleavage of DNA by topoisomerase II at different places: cleavage stimulated by amsacrine is consistent with the expected adenine requirement at position +1 whereas the predominant sites of DNA cleavage stimulated by MePyGA contain a cytosine at position +/- 1. This is the first instance where an anilinoacridine derivative differing only by the nature of the substituent at position 1' has been found to affect the catalytic activity of topoisomerase II differently. The spectroscopic and biochemical data lead to the conclusion that two functional domains can be identified in MePyGA: its anilino group can be regarded as a skeletal core to which are connected (i) the tricyclic acridine moiety which represents the DNA-binding domain and (ii) the N-methylpyrrole moiety which constitutes the topoisomerase II-targeted domain. The structure of the substituent at position 1' of the anilinoacridine chromophore evidently determines the location of the sites of DNA cleavage by topoisomerase II. These findings provide guidance for the synthesis and development of new topoisomerase II-targeted antitumor anilinoacridine derivatives.

A photoreactive analog of actinomycin (7-azidoactinomycin D) has been used in experiments to probe directly the shuffling hypothesis of Fox and Waring [Fox, K. R., & Waring, M. J. (1984) Nucleic Acids Res. 12, 9271-9285]. According to this theory, actinomycin D molecules initially interact with non-sequence-specific sites on DNA and subsequently "shuffle" along the polymer in a one-dimensional migratory fashion so as to locate their preferred sequence-dependent binding sites. In the study presented here, the drug-DNA complex was allowed to equilibrate (in the dark) for various periods of time, followed by photolysis which renders the complex irreversible and traps the ligand at its instantaneous binding sites. Visualization of the piperidine-labile sites and investigation of how the intensity of reaction at each site changes with time can provide direct confirmation of the shuffling hypothesis. The data reveal that actinomycin D does indeed engage in shuffling along the DNA. After only short equilibration times (20 s) actinomycin D is observed to bind to a variety of sites on the DNA, including many which are not regarded as canonical preferred binding sites at equilibrium. However, after longer periods of equilibration the intensity of reaction is shown to drop as a function of time at nonspecific sites, with corresponding increase at sequence-specific sites. In addition, base sequences which flank the intercalation sites can be seen to play a major role in influencing the binding and sequence specificity of actinomycin.

DNAase I footprinting has been used to probe the DNA sequence selectivity of the antitumor intercalating agents lucanthone (1), hycanthone (2), 6-chlorolucanthone (7), and four indazole analogs (IA-3-IA-6). The latter have a benzothiopyranoindazole chromophore substituted with a diethylaminoethyl side chain identical to that attached to the thioxanthenone chromophore of compounds 1, 2, and 7. IA-3 and IA-5 are lucanthone analogs bearing a methyl group at position 4, whereas IA-4 and IA-6 are hycanthone analogs bearing a hydroxymethyl group. IA-3 and IA-4 have an additional chloro group at position 6. Studies employing the 160-bp tyrT DNA fragment as substrate to assay inhibition of DNAase I-mediated cleavage show that both lucanthone and hycanthone bind preferentially to AT sites. They discriminate against GC-rich sequences as well as short runs of a single base, which are often cut more readily in the presence of the drugs compared to the control. The indazole analogs exhibit more pronounced selectivity of binding to AT sequences and promote enhanced DNAase I cleavage both at GC-rich sequences and at homooligomeric runs of adenines or thymines. The results of further DNAase I cleavage inhibition assays, performed with three more restriction fragments having different base pair arrangements, are fully consistent with those obtained with the tyrT fragment. They reveal that the preferred binding sequences for lucanthone, hycanthone, and the indazole analogs are predominantly composed of alternating A and T residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclease footprinting techniques have been employed to investigate the interaction between the diuretic drug amiloride, a sodium channel blocker with potential therapeutic use in the treatment of cystic fibrosis, and three DNA fragments of defined sequence. Using either DNAse I or micrococcal nuclease as probes, an unusual pattern of sequence-selective recognition of DNA has been detected. Amiloride binds selectively to sites rich in adenine and thymine residues, frequently with an apparent preference for 5'-TpX-3' steps, and discriminates strongly against GC-rich sequences which are sometimes cut more readily in the presence of the drug compared to the control. A detailed comparison with the actions of known selective DNA-binding antibiotics and drugs reveals a unique pattern of binding sites, different from those of typical intercalators on the one hand and those of minor groove-binders on the other. Amiloride is believed to adopt a pH-dependent tricyclic hydrogen-bonded conformation in solution which allows it to intercalate into DNA; consistent with this belief, we find that the footprinting pattern largely disappears at pH values above the pKa. Preliminary studies with three amiloride analogues have indicated the importance of two functional groups in the recognition of DNA. The possible relevance of selective DNA binding to activity in vivo is considered.

The protein transduction domain of HIV-1 TAT, TAT(48-60), is an efficient cell-penetrating peptide (CPP) that diffuses across the lipid membranes of cells despite eight cationic Arg and Lys residues. To understand its mechanism of membrane translocation against the free energy barrier, we have conducted solid-state NMR experiments to determine the site-specific conformation, dynamics, and lipid interaction of the TAT peptide in anionic lipid bilayers. We found that TAT(48-60) is a highly dynamic and nearly random-coil peptide in the lipid bilayer, and inserts into the membrane-water interface near the glycerol backbone region. Arg-phosphate salt bridge interaction was revealed by short guanidinium-phosphate distances and restricted dynamics of the guanidinium. Together with the observation of strong peptide-water cross peaks in 1H spin diffusion spectra, these results indicate that TAT binding to the membrane-water interface is stabilized not only by electrostatic attraction to the anionic lipids, but also by intermolecular hydrogen bonding with the lipid phosphates and water, which may take the role of intramolecular hydrogen bonds in canonical secondary structures. The random-coil structure of TAT and another CPP, penetratin, suggests that the lack of amphipathic structure is essential for rapid translocation of these Arg-rich CPPs across the lipid membrane without causing permanent damages to the membrane integrity.

We report the DNA binding properties of two hybrid molecules which result from the combination of the DNA sequence-specific minor groove ligand netropsin with the bithiazole moiety of the antitumor drug bleomycin. The drug-DNA interaction has been investigated by means of electric linear dichroism (ELD) spectroscopy and DNase I footprinting. In compound 1 the two moieties are linked by a flexible aliphatic tether while in compound 2 the two aromatic ring systems are directly coupled by a rigid peptide bond. The results are consistent with a model in which the netropsin moiety of compound 1 resides in the minor groove of DNA and where the appended bithiazole moiety is projected away from the DNA groove. This monocationic hybrid compound has a weak affinity for DNA and shows a strict preference for A and T stretches. ELD measurements indicate that in the presence of DNA compound 2 has an orientation typical of a minor groove binder. Similar orientation angles were measured for netropsin and compound 2. This ligand which has a biscationic nature tightly binds to DNA (Ka = 6.3 x 10(5) M-1) and is mainly an AT-specific groove binder. But, depending on the nature of the sequence flanking the AT site first targeted by its netropsin moiety, the bithiazole moiety of 2 can accommodate various types of nucleotide motifs with the exception of homooligomeric sequences. As evidenced by footprinting data, the bithiazole group of bleomycin acts as a DNA recognition element, offering opportunities to recognize GC bp-containing DNA sequences with apparently a preference (although not absolute) for a pyrimidine-G-pyrimidine motif. Thus, the bithiazole unit of bleomycin provides an additional anchor for DNA binding and is also capable of specifically recognizing particular DNA sequences when it is appended to a strongly sequence selective groove binding entity. Finally, a model which schematizes the binding of compound 2 to the sequence 5'-TATGC is proposed. This model readily explains the experimentally observed specificity of this netropsin-bithiazole conjugate.

DNase I footprinting has been used to probe the sequence selectivity of binding of a series of intercalating amsacrine-4-carboxamides and a related 9-aminoacridine-4-carboxamide to three DNA restriction fragments. These ligands have good experimental antileukemic activity, and for those members of the series that gave evaluable footprints, our principal finding is that they bind preferentially to GC-rich regions in agreement with the conclusion of equilibrium and kinetic measurements. The highest affinity sites generally occur in clusters of GC base pairs with runs of AT pairs being excluded from binding. It is important to appreciate that the 9-aminoacridine- and amsacrine-4-carboxamides exhibit a very high degree of selectivity for GC sites which, to our knowledge, has not been previously matched by acridine derivatives in footprinting experiments. The principal determinant of specificity appears to be the 4-carboxamide group itself since neither variations in the terminal funtionality of the 4-carboxamide sidechain nor the presence of the 9-anilino substituent modifies sequence preferences. The molecular origins of selectivity may be discerned in terms of potential hydrogen bonding interactions between the 4-carboxamide moiety and carbonyl oxygen and amino groups of GC base pairs in the DNA minor groove at CG dinucleotide sites. The related therapeutic agent amsacrine failed to inhibit cleavage by DNase I, so no conclusion can be drawn concerning its binding selectivity, save to note that amsacrine does not possess the 4-carboxamide group which appears to be the crucial determinant of GC specificity. Whether selectivity for binding to GC-rich sequences is an important element in the antitumor activity of both the 9-aminoacridine- and amsacrine-4-carboxamides remains to be determined.

Tachyplesin-I (TP-I) is a 17-residue beta-hairpin antimicrobial peptide containing two disulfide bonds. Linear analogs of TP-I where the four Cys residues were replaced by aromatic and aliphatic residues, TPX4, were found to have varying degrees of activities, with the aromatic analogs similarly potent as TP-I. Understanding the different activities of the linear analogs should give insight into the mechanism of action of TP-I. To this end, we have investigated the dynamic structures of the active TPF4 and the inactive TPA4 in bacteria-mimetic anionic POPE/POPG bilayers and compared them with the wild-type TP-I using solid-state NMR spectroscopy. 13C isotropic chemical shifts and backbone (phi, psi) torsion angles indicate that both TPF4 and TPA4 adopt beta-strand conformations without a beta-turn at key residues. 1H spin diffusion from lipid chains to the peptide indicates that the inactive TPA4 binds to the membrane-water interface, similar to the active TP-I. Thus, neither the conformation nor the depth of insertion of the three peptides correlates with their antimicrobial activities. In contrast, the mobility of the three peptides correlates well with their activities: the active TP-I and TPF4 are both highly mobile in the liquid-crystalline phase of the membrane while the inactive TPA4 is completely immobilized. The different mobilities are manifested in the temperature-dependent 13C and 15N spectra, 13C-1H and 15N-1H dipolar couplings and 1H rotating-frame spin-lattice relaxation times. The dynamics of TP-I and TPF4 are both segmental and global. Combined, these data suggest that TP-I and TPF4 disrupt the membrane by large-amplitude motion in the plane of the membrane. The loss of this motion in TPA4 due to aggregation significantly weakens its activity because a higher peptide concentration is required to disturb lipid packing. Thus molecular motion, rather than structure, appears to be the key determinant for the membrane-disruptive activities of tachyplesins.

This paper is focused on the thermodynamics and the structural investigation of the interaction of the antimicrobial peptide dicynthaurin monomer with model lipid membranes composed of mixtures of 1-palmitoyl-2-oleyl-glycerophosphocholine and -glycerophosphoglycerol. The thermodynamic binding parameters as obtained by isothermal titration calorimetry reveal strong binding toward the lipid model system dominated by large chemical binding constants which exceeds the electrostatic binding effects and thus suggests insertion of the amphipathic alpha-helical peptide into the hydrophobic membrane core. Circular dichroism study shows that the peptide exhibits trans-membrane alpha-helix secondary structure. Neutron diffraction measurements using partially deuterated sequences were successfully applied to determine the orientation of the peptide thus proving insertion into the hydrophobic membrane core. This insertion and the formation of higher order porelike aggregates is assumed to be the most relevant event in microbial membrane perturbation that in vivo finally leads to bacterial cell death on a fast time scale.

The conformation and membrane topology of the disulfide-stabilized antimicrobial peptide tachyplesin I (TP) in lipid bilayers are determined by solid-state NMR spectroscopy. The backbone (phi and psi) torsion angles of Val(6) are found to be -133 degrees and 142 degrees , respectively, and the Val(6) CO-Phe(8) H(N) distance is 4.6 A. These constrain the middle of the N-terminal strand to a relatively ideal antiparallel beta-sheet conformation. In contrast, the phi angle of Gly(10) is +/-85 degrees , consistent with a beta-turn conformation. Thus, TP adopts a beta-hairpin conformation with straight strands, similar to its structure in aqueous solution but different from a recently reported structure in DPC micelles where bending of the two beta-strands was observed. The Val(6) and Gly(10) CO groups are both 6.8 A from the lipid (31)P, while the Val(6) side chain is in (1)H spin diffusion contact with the lipid acyl chains. These results suggest that TP is immersed in the glycerol backbone region of the membrane and is oriented roughly parallel to the plane of the membrane. This depth of insertion and orientation differs from those of the analogous beta-sheet antimicrobial peptide protegrin-1 and suggest the importance of structural amphiphilicity in determining the location and orientation of membrane peptides in lipid bilayers.

Although the membrane-associated surfactant protein B (SP-B) is an essential component of lung surfactant, which is itself essential for life, the molecular basis for its activity is not understood. SP-B's biophysical functions can be partially mimicked by subfragments of the protein, including the C-terminus. We have used NMR to determine the structure of a C-terminal fragment of human SP-B that includes residues 63-78. Structure determination was performed both in the fluorinated alcohol hexafluoro-2-propanol (HFIP) and in sodium dodecyl sulfate (SDS) micelles. In both solvents, residues 68-78 take on an amphipathic helical structure, in agreement with predictions made by comparison to homologous saposin family proteins. In HFIP, the five N-terminal residues of the peptide are largely unstructured, while in SDS micelles, these residues take on a well-defined compact conformation. Differences in helical residue side chain positioning between the two solvents were also found, with better agreement between the structures for the hydrophobic face than the hydrophilic face. A paramagnetic probe was used to investigate the position of the peptide within the SDS micelles and indicated that the peptide is located at the water interface with the hydrophobic face of the helix oriented inward, the hydrophilic face of the helix oriented outward, and the N-terminal residues even farther from the micelle center than those on the hydrophilic face of the alpha-helix. Interactions of basic residues of SP-B with anionic lipid headgroups are known to have an impact on function, and these studies demonstrate structural ramifications of such interactions via the differences observed between the peptide structures determined in HFIP and SDS.

RTD-1 is a cyclic beta-hairpin antimicrobial peptide isolated from rhesus macaque leukocytes. Using (31)P, (2)H, (13)C, and (15)N solid-state NMR, we investigated the interaction of RTD-1 with lipid bilayers of different compositions. (31)P and (2)H NMR of uniaxially oriented membranes provided valuable information about how RTD-1 affects the static and dynamic disorder of the bilayer. Toward phosphatidylcholine (PC) bilayers, RTD-1 causes moderate orientational disorder independent of the bilayer thickness, suggesting that RTD-1 binds to the surface of PC bilayers without perturbing its hydrophobic core. Addition of cholesterol to the POPC membrane does not affect the orientational disorder. In contrast, binding of RTD-1 to anionic bilayers containing PC and phosphatidylglycerol lipids induces much greater orientational disorder without affecting the dynamic disorder of the membrane. These correlate with the selectivity of RTD-1 for anionic bacterial membranes as opposed to cholesterol-rich zwitterionic mammalian membranes. Line shape simulations indicate that RTD-1 induces the formation of micrometer-diameter lipid cylinders in anionic membranes. The curvature stress induced by RTD-1 may underlie the antimicrobial activity of RTD-1. (13)C and (15)N anisotropic chemical shifts of RTD-1 in oriented PC bilayers indicate that the peptide adopts a distribution of orientations relative to the magnetic field. This is most likely due to a small fraction of lipid cylinders that change the RTD-1 orientation with respect to the magnetic field. Membrane-bound RTD-1 exhibits narrow line widths in magic-angle spinning spectra, but the sideband intensities indicate rigid-limit anisotropies. These suggest that RTD-1 has a well-defined secondary structure and is likely aggregated in the membrane. These structural and dynamical features of RTD-1 differ significantly from those of PG-1, a related beta-hairpin antimicrobial peptide.

The dynamics and aggregation of a beta-sheet antimicrobial peptide, protegrin-1 (PG-1), are investigated using solid-state NMR spectroscopy. Chemical shift anisotropies of F12 and V16 carbonyl carbons are uniaxially averaged in 1,2-dilauryl-sn-glycero-3-phosphatidylcholine (DLPC) bilayers but approach rigid-limit values in the thicker 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC) bilayers. The Calpha-Halpha dipolar coupling of L5 is scaled by a factor of 0.16 in DLPC bilayers but has a near-unity order parameter of 0.96 in POPC bilayers. The larger couplings of PG-1 in POPC bilayers indicate immobilization of the peptide, suggesting that PG-1 forms oligomeric aggregates at the biologically relevant bilayer thickness. Exchange NMR experiments on F12 (13)CO-labeled PG-1 show that the peptide undergoes slow reorientation with a correlation time of 0.7 +/- 0.2 s in POPC bilayers. This long correlation time suggests that in addition to aggregation, geometric constraints in the membrane may also contribute to PG-1 immobilization. The PG-1 aggregates contact both the surface and the hydrophobic center of the POPC bilayer, as determined by (1)H spin-diffusion measurements. Thus, solid-state NMR provides a wide range of information about the molecular details of membrane peptide immobilization and aggregation in lipid bilayers.

We study the interaction of antimicrobial peptides with lipopolysaccharide (LPS) bilayers to understand how antimicrobial peptides interact with the LPS monolayer on the outer membrane of Gram-negative bacteria. LPS in water spontaneously forms a multilamellar structure composed of symmetric bilayers. We performed X-ray lamellar diffraction and wide-angle in-plane scattering to study the physical characteristics of LPS multilayers. The multilayer alignment of LPS is comparable to phospholipids. Thus, it is suitable for the application of oriented circular dichroism (OCD) to study the state of peptides in LPS bilayers. At high hydration levels, the chain melting temperature in multilamella detected by X-ray diffraction is the same as that of LPS aqueous dispersions, as measured by calorimetry. LPS has a strong CD, but with a careful subtraction of the lipid background, the OCD of peptides in LPS is measurable. The method was tested successfully with melittin. It was then applied to two representative antimicrobial peptides, magainin and protegrin. At peptide concentrations comparable to the physiological conditions, both peptides penetrate transmembrane in LPS bilayers. The results imply that antimicrobial peptides readily penetrate the LPS monolayer of the outer membrane.

RTD-1 is a recently discovered cyclic peptide that, like other well-studied antimicrobial peptides, appears to bind to the lipid matrix of cell membrane in the initial stage of activity. We studied the states of RTD-1 bound to lipid bilayers by two methods: oriented circular dichroism and X-ray diffraction. RTD-1 shows two physically distinct bound states in lipid bilayers like magainins, protegrins, alamethicin, and melittin that were previously studied. However, the nature of transition between the two states is different for RTD-1 as compared with the aforementioned peptides. In one of the two states, RTD-1 is oriented with its backbone ring parallel to the plane of the bilayer. Only in this state RTD-1 induces membrane thinning. But the effect of membrane thinning is much weaker than all other peptides, suggesting that the mechanism of RTD-1 may be different from the other peptides.

The antitumor drug ditercalinium is a rare example of a noncovalent DNA-binding ligand that forms bisintercalation complexes via the major groove of the double helix. Previous structural studies have revealed that the two connected pyridocarbazolium chromophores intercalate into DNA with the positively charged bis(ethylpiperidinium) linking chain oriented to the wide groove side of the helix. Although the interaction of ditercalinium with short oligonucleotides containing 4-6 contiguous GC base pairs has been examined in detail by biophysical and theoretical approaches, the sequence preference for ditercalinium binding to long DNA fragments that offer a wide variety of binding sites has been investigated only superficially. Here we have investigated both sequence preferences and possible molecular determinants of selectivity in the binding of ditercalinium to DNA, primarily using methods based upon DNase I footprinting. A range of multisite DNA substrates, including several natural restriction fragments and different PCR-generated fragments containing unconventional bases (2,6-diaminopurine, inosine, uridine, 5-fluoro- and 5-methylcytosine, 7-deazaguanine, 7-deazaadenine, and N(7)-cyanoboranoguanine), have been employed to show that ditercalinium selectively recognizes certain GC-rich sequences in DNA and to identify some of the factors which affect its DNA-binding sequence selectivity. Specifically, the footprinting data have revealed that the 2-amino group on the purines or the 5-methyl group on the pyrimidines is not essential for the formation of ditercalinium-DNA complexes whereas the major groove-oriented N(7) of guanine does appear as a key element in the molecular recognition process. The loss of N(7) at guanines but not adenines is sufficient to practically abolish sequence-selective binding of ditercalinium to DNA. Thus, as expected for a major groove binding drug, the N(7) of guanine is normally required for effective complex formation with GC base pairs, but interestingly the substitution of the N(7) with a relatively bulky cyanoborane group does not markedly affect the sequence recognition process. Therefore, the hydrogen bond accepting capability at N(7) of guanines is not sufficient to explain the GC-selective drug-DNA association, and the implications of these findings are considered.

The effects of a wide range of DNA binding drugs on peptide nucleic acid (PNA) binding to double-stranded DNA by strand displacement have been investigated using a gel retardation assay. The bis-PNA [H-(Lys)-TTJTTJTTTT-(eg)(3)-TTTTCTTCTT-Lys-NH(2)] was used together with a 248 bp DNA fragment containing an appropriate target for the PNA. Most of the ligands that were studied, including DNA minor groove binders as well as intercalators and bis-intercalators, either have no effect or strongly inhibit PNA binding to DNA. By contrast, quinoxaline antibiotics facilitate PNA-DNA complex formation. The "PNA-helper" effect of echinomycin was studied in more detail using time and temperature dependence experiments to elucidate the mechanism. PNA binding to DNA follows pseudo-first-order kinetics, but the initial rate of binding is accelerated more than 10-fold in the presence of 10 microM echinomycin. The activation energy for PNA binding to dsDNA is lowered 2-fold by the antibiotic (45 vs 90 kJ/mol in the control). The reasons why quinoxalines promote the binding of PNA to DNA are not entirely clear but may well include distortions (opening) of the double helix that facilitate PNA invasion. This study establishes that the efficacy of DNA-targeted PNA antigene molecules could potentially be enhanced by judiciously adding certain DNA-interactive ligands.

Protegrin-1 (PG-1), a beta-sheet antimicrobial peptide, was studied in aligned lipid bilayers by oriented circular dichroism (OCD). All of its spectra measured in a variety of lipid compositions were linear superpositions of two primary basis spectra, indicating that PG-1 existed in two different states in membranes. We designated these as state S and state I. The state assumed by PG-1 was strongly influenced by lipid composition, peptide concentration, and hydration condition. We have previously reported that the helical peptides, alamethicin and magainin, also exhibit two distinct OCD basis spectra-one corresponding to surface adsorption with the helix parallel to the bilayer and the other with perpendicular transbilayer insertion. States S and I of PG-1 may correspond to the surface state and the insertion state of alamethicin, since they show a similar dependence on lipid composition, peptide concentration, and hydration condition. Nonoriented CD spectra obtained from vesicle, micelle, and solution preparations are not linear superpositions of the basis spectra of the states S and I. This indicates that a molecular orientation change alone is insufficient to describe the S left and right arrow I transition. Rather, a more complicated process is taking place, perhaps involving a change in the hydrogen bonding pattern of the backbone. Although the structural basis of the OCD spectra remains to be determined, the discovery of two distinct states can provide information about dynamic changes of PG-1 in membranelike environments, properties undoubtedly related to its antimicrobial and cytotoxic effects.

In order to investigate the possible importance of Hoogsteen base pairing to the DNA-binding ability of echinomycin, quantitative DNase I footprinting has been performed. The substrate was the tyrT DNA restriction fragment, either "native" or substituted with one of the purine analogs 2'-deoxy-7-deazaadenosine and 2'-deoxy-7-deazaguanosine in both strands. The modified DNA species were prepared by PCR and selectively labeled at the 5' terminus of one strand (usually the upper "Watson" strand) with [32P]ATP and polynucleotide kinase. Proper incorporation of the analog nucleotides was verified by Maxam-Gilbert G- and C-sequencing reactions as well as exposure to osmium tetroxide and diethyl pyrocarbonate. OsO4 was found to react strongly with the 7-deaza nucleotides, providing a good check of faithful incorporation. The previously observed echinomycin-induced hyperreactivity of purines toward diethyl pyrocarbonate was eliminated by incorporating the appropriate 7-deazapurine. The DNase I footprinting titration studies greatly refined the existing knowledge of the DNA-binding characteristics of echinomycin, as they revealed five general types of concentration-dependent behavior at single-bond resolution. Estimates of microscopic binding constants at individual DNA binding sites were obtained by measuring the antibiotic concentration which produced a half-maximal effect on the concentration of a given DNase I cleavage product. All binding sites contained one or more CpG steps, and all CpG steps analyzed formed part of a binding site for echinomycin. No consistent differences in the estimated binding constants for these sites were observed by comparing normal and modified DNAs, indicating that the abolition of formal Hoogsteen pairs did not significantly alter the thermodynamics of echinomycin-DNA interaction. The lack of any detectable decrease in binding constants for critical sites in the 7-deazapurine-substituted DNAs argues against any anti-syn conformational transition of purine nucleosides occurring in association with the bis-intercalative complex formation.

A bifunctional molecule in which an ellipticine chromophore is attached to a distamycin residue via a diaminopropyl tether has been designed and synthesized in the expectation of creating a hybrid molecule capable of bidentate binding to DNA by both intercalation and minor-groove interactions. The strength and mode of binding to DNA of this conjugate have been studied by means of circular and linear dichroism as well as by stopped-flow kinetics and measurements of reactivity toward a chemical probe. The results converge to reveal that the ellipticine moiety of the hybrid largely dominates the binding reaction with DNA. In the presence of chromatin, the hybrid molecule binds preferentially to the internucleosomal DNA, a preference dictated by its intercalating chromophore. Theoretical computations were performed on the comparative complexation energies of distamycin, the ellipticine derivative, and the hybrid ligand with a B-representative octanucleotide, d(GCATATGC)2. The best binding configuration of the ellipticine derivative locates its aminoalkyl side chain in the minor groove where distamycin is also present. The molecular modeling analysis fully supports the involvement of a bimodal binding process for the hybrid and reveals that the binding of the conjugate to DNA favors a pronounced bending toward the minor groove. This effect is attributed to intercalation of the ellipticine chromophore. An interesting link is established between the DEPC reactivity experiments and the theoretical computations, suggesting that DEPC can be used as a probe for drug-induced DNA bending. On the basis of these results, we propose the design of a new hybrid ligand bearing an additional positively-charged amidine side chain to confer higher DNA-binding affinity.

The polymeric DNA and model duplex oligonucleotide complexes of the bisquinoline analogue of echinomycin (2QN) have been studied by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy, with the quinoline chromophores of the drug used as intrinsic probes. Plots of ODMR transition frequencies versus monitored wavelength revealed heterogeneity in the phosphorescence emission of 2QN which was ascribed to the presence of a major and minor conformation of the drug in aqueous solutions (referred to as the red and blue forms of 2QN, respectively, in this report). ODMR results, in conjunction with findings from low-temperature phosphorescence investigations, indicate that the quinoline chromophores of the major (red) form of 2QN are involved in aromatic stacking interactions in complexes with the natural DNAs from Escherichia coli, Micrococcus lysodeikticus, Clostridium perfringens, and calf thymus as evidenced by red shifts in the phosphorescence 0,0-band of the drug, reductions in the phosphorescence lifetime and zero-field splitting (zfs) D and E parameters, and polarity reversals of the ODMR slow passage signals upon complex formation between the analogue and DNA. The polarity reversals, which reflect shifts in the triplet-state sublevel populations induced by complex formation, apparently result from changes in the triplet sublevel decay constants upon binding to the natural DNAs. The 2QN complexes of the double-stranded alternating copolymers poly(dG-dC).poly(dG-dC) [abbreviated as poly[d(G-C)2]] and poly(dA-dT).poly(dA-dT) [abbreviated as poly(dA-dT).poly(dA-dT) [abbreviated as poly[d(A-T)2], the homopolymer duplexes poly(dG).poly(dC) [abbreviated as poly(dG.dC)] and poly(dA).poly(dT) [abbreviated as poly(dA.dT)], and the self-complementary oligonucleotides d(ACGT)2, d(TCGA)2, and d(ACGTACGT)2 were also investigated. The extent of reduction of the zfs D parameter (delta D) for the major form of 2QN upon complex formation with the polymeric DNAs was found to scale linearly with the standard free energy of the drug-DNA interaction (delta G degrees) calculated from previously reported binding studies for these targets [Fox, K. R., et al. (1980) Biochem. J. 191, 729-740]. This relationship between spectroscopic and thermodynamic properties of the 2QN-polynucleotide complexes is a consequence of the effects of base stacking interactions on the electronic states of the intercalator, which were postulated to arise from second-order shifts of the ground-state and the triplet-state energies of the complex on the basis of a modification of the solvent effect theory of van Egmond et al. [(1975) Chem. Phys. Lett. 34, 423-426].

Results from a high-resolution deoxyribonuclease I (DNase I) footprinting titration procedure are described that identify preferred daunomycin binding sites within the 160 bp tyr T DNA fragment. We have obtained single-bond resolution at 65 of the 160 potential binding sites within the tyr T fragment and have examined the effect of 0-3.0 microM total daunomycin concentration on the susceptibility of these sites toward digestion by DNase I. Four types of behavior are observed: (i) protection from DNase I cleavage; (ii) protection, but only after reaching a critical total daunomycin concentration; (iii) enhanced cleavage; (iv) no effect of added drug. Ten sites were identified as the most strongly protected on the basis of the magnitude of the reduction of their digestion product band areas in the presence of daunomycin. These were identified as the preferred daunomycin binding sites. Seven of these 10 sites are found at the end of the triplet sequences 5'ATGC and 5'ATCG, where the notation AT indicates that either A or T may occupy the position. The remaining three strongly protected sites are found at the ends of the triplet sequence 5'ATCAT. Of the preferred daunomycin binding sites we identify in this study, the sequence 5'ATCG is consistent with the specificity predicted by the theoretical studies of Chen et al. [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466] and is the very sequence to which daunomycin is observed to be bound in two recent X-ray crystallographic studies. Solution studies, theoretical studies, and crystallographic studies have thus converged to provide a consistent and coherent picture of the sequence preference of this important anticancer antibiotic.

The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.

An homologous series of diacridines containing two 9-aminoacridine chromophores linked via a simple methylene chain has been studied in order to investigate the minimum interchromophore separation required to permit bifunctional intercalation. Viscometric, sedimentation, and electric dichroism experiments show that compounds having one to four methylene groups in the linker are restricted to monofunctional intercalation, whereas the interaction becomes bifunctional when the chain length is increased to six carbons or more. The results indicate that bifunctional reaction occurs with an interchromophore distance not exceeding 8.8 A, implying that intercalation by these compounds is not subject to neighbor exclusion if the mode of binding is of the classical intercalation type.

Sedimentation and viscosity experiments reveal that when crystal violet binds to closed circular deoxyribo- nucleic acid (DNA) the helix becomes unwound. The average angle of unwinding per bound dye molecule is 9.8 f 0.6'. Viscosity measurements with sonicated rodlike fragments of calf thymus DNA provide evidence that the binding causes an apparent decrease in contour length. Scatchard plots for binding to DNA are curvilinear, concave upward, indicative of strong exclusion effects and/or heterogeneity of binding sites having different affinity. By contrast, binding curves for RNA are curvilinear, concave downward, suggestive of cooperativity. Crystal violet binds preferentially to DNA, its intrinsic asso- ciation constant being 15-fold lower for ribonucleic acid (RNA) at ionic strength 0.01 and 46-fold lower in 0.2 M salt solution. Heat denaturing DNA radically alters the binding mechanism such that Scatchard plots acquire the "humped" appearance characteristic of a cooperative reaction. Its preference for binding to helical DNA is confirmed by a large increase in the "~elting" temperature of calf thymus DNA in buffer of low ionic strength. Raising the ionic strength Studies of the interaction between triphenylmethane dyes and deoxyribonucleic acid (DNA) have produced conflicting accounts of the mechanism of binding (Lerman, 1961, 1964 a,b; Neville & Davies, 1966; Armstrong & Panzer, 1972; Muller & Gautier, 1975). Lerman (1961, 1964a) reported that these dyes increase the viscosity of DNA to an extent comparable to that found for acridines, which led him to propose that both types of ligand intercalate. He also found that on binding to DNA there was a marked decrease in the reactivity of the amino groups of pararosaniline; this he offered as further evidence that the cation is at least partially buried within the helix (Lerman, 1964b). Armstrong & Panzer (1972) confirmed the increase in viscosity up to r (moles of dye bound per mole of DNA phosphate) = 0.1 and suggested that pararosaniline intercalates so as to exclude similar binding at the three nearest-neighbor sites on either side of a bound dye molecule. In addition, they identified a second form of binding at r > 0.125 which did not affect viscosity and which they postulated to involve external attachment to the surface of the DNA helix. In contrast to these reports, X-ray dif- fraction studies on pararosaniline-DNA fibers did not reveal decreases affinity constants generally, and at a salt concen- tration of 0.75 M binding to secondary sites on DNA is sub- stantially reduced. Temperature-jump measurements reveal two fast closely coupled relaxation times at low levels of binding whereas at higher binding ratios an additional two exponential components can be resolved, again closely coupled but well separated from the fast effects. Thus, the ligand forms at least two distinct complexes with DNA at low levels of binding, and there are not less than four bound species at higher binding ratios. The visible absorption spectrum of the crystal violet-DNA complex becomes increasingly batho- chromic and hypochromic as the binding ratio is raised. However, the spectrum of the dye bound to RNA is charac- terized by a strong hypsochromic component and a much greater hypochromism at higher binding ratios than is seen with DNA. These spectral properties are attributed to changes in the propeller-like conformation of the dye due to rotations which affect the relative disposition of the phenyl rings and/or their dimethylamino substitutents.

Four DNA restriction fragments, designated tyrT, pTyr2, pUC13, and Xbs1, have been used as substrates for footprinting studies with DNase I in the presence of the anthracycline antibiotic nogalamycin. With each fragment a distinct pattern of antibiotic-protected binding sites is observed, but no concensus sequence emerges from the data. All sites are located in regions of alternating purine-pyrimidine sequence, most commonly associated with the dinucleotide steps TpG (CpA) and GpT (ApC), suggesting that the preferred binding sites may contain all four nucleotides and/or that peculiarities of the dynamics of DNA conformation at alternating sequences may be critical for nogalamycin binding. Some concentration dependence of footprinting patterns is evident, in contrast to previous studies with a variety of sequence-specific ligands. Enhanced susceptibility to attack by DNase I is commonly observed at sequences flanking strong antibiotic-binding sites. Nogalamycin selectively inhibits cleavage of DNA at certain guanine-containing sequences by the G-specific photosensitized reaction with methylene blue. Comparison of these effects with its action on the G-specific reaction with dimethyl sulfate suggests that the amino sugar moiety of nogalamycin may be preferentially located in the minor helical groove at some binding sites but in the major groove at others.

The kinetics of association between the quinoxaline antitumor antibiotic echinomycin and DNA have been studied by stopped-flow methods. With natural DNAs, the reaction profile is completely described by a single exponential, the time constant for which varies linearly with the DNA concentration. This bimolecular rate constant is similar for both calf thymus and Micrococcus lysodeikticus DNA (k = 6 X 10(4) M-1 s-1 at 25 degrees C, I = 0.01) and is probably dominated by interaction with relatively weak but abundant binding sites from which the antibiotic dissociates fairly quickly. The observed single exponential suggests a molecular mechanism of binding in which both chromophores of the antibiotic become intercalated simultaneously rather than sequentially; no transition from a mono-intercalated state to a bis-intercalated state could be detected. The reaction is slowed by a factor of about 3 on raising the salt concentration from I = 0.01 to I = 0.5. Binding to poly(dA-dT) is also described by a single exponential, the time constant for which is about 3 times faster than that seen with natural DNAs. By contrast, the interaction with poly-(dG-dC) requires two exponentials for a proper description, the faster of which is similar to that seen with natural DNAs. This may reflect the initial interaction of the antibiotic with two types of sequences, tentatively identified as GpC and CpG, from which it dissociates at very different rates. The differences in kinetic behavior may be explicable on the basis of an alternating B structure for poly(dA-dT) and a more classical B form for poly(dG-dC).

Sedimentation experiments with closed circular duplex PM2 deoxyribonucleic acid (DNA) and viscosity measurements with sonicated rodlike DNA fragments have been performed to investigate unwinding and extension of the DNA helix associated with binding of homologous series of diacridines. When the acridine nuclei are linked via 9-amino substituents with chains permitting interchromophore separation in the critical region between 8.8 and 11.3 A, it is found that substituents at positions 2, 3, and 6 restrict bifunctional intercalation to compounds capable of sandwiching at least two base pairs in the intercalated complex. Unsubstituted diacridines, and derivatives bearing an ethyl group at position 4, can bis-intercalate with a significantly shorter linking chain. The presence of amino groups in the linker does not affect the ability to bis-intercalate but increases the helix unwinding angle by 30--40% and decreases the helix extension per bound ligand molecule by 11--18%. Bifunctional reaction is observed with a diacridine linked via the 4--4' positions showing that 9-aminoacridine can intercalate in at least two major orientations with the 9 or 10 position directed toward the helix axis. Both orientations can be accommodated simultaneously within a single diacridine linked via the 9--4' positions with a bridge sufficiently long to encompass two base pairs. An acridone substituted at positions 4 and 5 with positively charged side chains intercalates with a helix unwinding angle indistinguishable from that of ethidium whereas an uncharged bisacridone failed to yield a detectable complex with DNA. Tilorone dihydrochloride, a structurally similar antiviral drug, was found to intercalate with an unwinding angle approximately half that of ethidium. The potentially bifunctional bisquinoline drug dequalinium causes relaxation of the supercoiling of PM2 DNA but probably not by simultaneous intercalation of both its chromophores.