The B-ring substituent at C-7 of colchicine and the alpha-C-terminus of tubulin communicate through the "tail-body" interaction

The carboxy terminals of alphabeta-tubulins are flexible regions rich in acidic amino acid residues that play an inhibitory role in the polymerization of tubulin to microtubules. We have shown that the binding of colchicine and its B-ring analogs (with C-7 substituents) to tubulin are pH sensitive and have high activation energies. Under identical conditions, the binding of analogs without C-7 substituents is pH independent and has lower activation energy. Beta-C-terminus-truncated tubulin (alphabeta(s)) shows similar pH sensitivity and activation energy to native tubulin (alphabeta). Removal of the C-termini of both subunits of tubulin (alpha(s)beta(s)) or the binding of a basic peptide P2 to the negatively charged alpha-C-terminus of tubulin causes a colchicine-tubulin interaction independent of pH with a low activation energy. Tubulin dimer structure shows that the C-terminal alpha-tail is too far from the colchicine binding site to interact directly with the bound colchicine. Therefore, it is likely that the interaction of the alpha-C-terminus with the main body of tubulin indirectly affects the colchicine-tubulin interaction via conformational changes in the main body. We therefore conclude that in the presence of tail-body interaction, a B-ring substituent makes contact with the alpha-tubulin and induces significant conformational changes in alpha-tubulin.     

1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin.

A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.     

The interaction of the B-ring of colchicine with alpha-tubulin: a novel footprinting approach

Tubulin, the major structural component of the microtubules, participates actively in mitotic spindle formation and chromosomal organization during cell division. Tubulin is the major target for a variety of anti-mitotic drugs. Some of the drugs, such as Vinca alkaloids and taxol, are routinely used for cancer chemotherapy. It is unfortunate that our knowledge of the binding sites on tubulin of these drugs is limited because of lack of a useful and appropriate tool. The photoaffinity labeling approach is the major technique available at present to detect the binding sites of drugs on tubulin. This method, however, has several limitations. First, only part of the binding site can be identified, namely, the residues which react with the photoaffinity label. Second, there are regions of tubulin which are not at the binding site but are affected by the binding of the drug; these regions can not be detected by the photoaffinity labeling approach. The third, and perhaps most serious, limitation is that the traditional approach can detect areas which have nothing to do with the binding of the ligand but which are within a certain distance of the binding site, that distance being less than the length of the photoreactive moiety attached to the ligand. There has been a great deal of controversy on the localization of the binding site of colchicine on tubulin, with some reports suggesting that the binding site is on alpha and some supporting a binding site on beta. Colchicine also has significant effects on tubulin conformation, but the regions which are affected have not been identified. We have attempted here to address these questions by a novel "footprinting" method by which the drug-binding sites and as well as the domain of tubulin affected by drug-induced conformational changes could be determined. Here, we report for the first time that the interaction of the B-ring of colchicine with the alpha-subunit affects a domain of tubulin which appears to be far from its binding site. This domain includes the cysteine residues at positions 295, 305, 315 and 316 on alpha-tubulin; these residues are located well away from the alpha/beta interface where colchicine appears to bind. This is correlated with the stabilizing effect of colchicine on the tubulin molecule. Furthermore, we also found that the B-ring of colchicine plays a major role in the stability of tubulin while the A and the C-rings have little effect on it. Our results therefore, support a model whereby colchicine binds at the alpha/beta interface of tubulin with the B-ring on the alpha-subunit and the A and the C-rings on the beta-subunit.     

NMR and molecular modeling characterization of RGD containing peptides.

The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d6 of the cyclic pentapeptides, [formula: see text], a potent fibrinogen receptor antagonist, and [formula: see text], a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. 1H-1H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from 3JNH-H alpha coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.     

Solution conformation of endothelin and point mutants by nuclear magnetic resonance spectroscopy.

Two-dimensional NMR techniques were utilized to determine the secondary structural elements of endothelin-1 (ET-1), a potent vasoconstrictor peptide, and two of its point mutants, Met-7 to Ala-7 (ETM7A), and Asp-8 to Ala-8 (ETD8A) in acetic acid-d3/water solution. Sequence specific NMR assignments were determined for all three peptides, as well as chemical shifts and NOE connectivity patterns. The chemical shifts of ET-1 and ETM7A are identical (+/- 0.05 ppm) except for the site of substitution, whereas marked shift changes were detected between ET-1 and ETD8A. These chemical shift differences imply that the Asp-8 to Ala-8 mutation has induced a conformational change relative to the parent conformation. All three molecules show the same basic nuclear Overhauser effect (NOE) pattern, which suggests that the gross conformation of all three molecules is the same. Small changes in sequential NOE intensities and changes in medium-range NOE patterns indicate that there are subtle conformational differences between ET-1 and ETD8A.     

Conformation and dynamics of an Fab'-bound peptide by isotope-edited NMR spectroscopy.

The dynamics and conformation of the peptide antigen MHKDFLEKIGGL bound to the Fab' fragment of the monoclonal antipeptide antibody B13A2, raised against a peptide from myohemerythrin, have been investigated by isotope-edited NMR techniques. The peptides were labeled with 15N (98%) or 13C (99%) at the backbone of individual amino acid residues. Well-resolved amide proton and nitrogen backbone resonances were obtained and assigned for eight of the 12 residues of this bound peptide. Significant resonance line width and chemical shift differences were observed. The 15N and 1H line width variations are attributed to differential backbone mobilities among the bound peptide residues which are consistent with the previously mapped epitope of this peptide antigen. Local structural information was obtained from isotope-directed NOE studies. The approximate distances associated with the experimental NOEs were estimated on the basis of a theoretical NOE analysis involving the relative integrated intensities of the NOE and source peaks. In this way, the sequential NH-NH NOEs obtained for seven of the Fab'-bound peptide residues were shown to correspond to interproton separations of approximately 3 A or less. Such short distances indicate that the backbone dihedral angles of these residues are in the alpha rather than the beta region of phi,psi conformational space; the peptide most likely adopts a helical conformation from F5 to G11 within the antibody combining site. The significance of these results with respect to the type and extent of conformational information obtainable from studies of high molecular weight systems is discussed.     

Solution structure of a peptide derived from the oncogenic protein beta-Catenin in its phosphorylated and nonphosphorylated states

Beta-Catenin plays an essential role in the Wingless/Wnt signaling cascade. Phosphorylation of beta-Catenin in its N-terminal region by the kinase GSK-3beta is required for the interaction with the SCF-beta-TrCP protein complex that targets beta-Catenin for proteasome degradation. In the present work, we used two peptides of 32 amino acids referred to beta-Cat17-48 and P-beta-Cat17-48 for the phosphorylated peptide at the two sites Ser33 and Ser37. Circular dichroism and NMR techniques were used to assess the influence of the phosphorylation. The spectra of the peptides at pH 7.2 were completely assigned. Analysis of the medium-range NOE connectivities indicated that beta-Cat17-48 seems to be only poorly folded. These data are in agreement with the result of structure calculations. P-beta-Cat17-48 possesses two helical segments around the DpSGXXpS motif, which forms a large bent with the phosphate groups pointing out of the structure. On the contrary, beta-Cat17-48 shows less well-defined secondary structures and appears as a more flexible peptide, but adopts in the motif DSGXXS a more compact conformation than P-beta-Cat17-48. Differences in this molecular region suggest that conformational changes of phosphorylated beta-Catenin play an important role for the interaction with the SCF-beta-TrCP protein complex.     

Solution structure of endothelin-3 determined using NMR spectroscopy.

The aqueous solution structure of the 21-residue vasoactive peptide hormone endothelin-3 has been determined using high-resolution NMR spectroscopy. A total of 177 proton-proton distance measurements and 5 chi 1 dihedral angle constraints derived from NMR spectra were used to calculate the structure using a combination of distance geometry and dynamical simulated annealing calculations. The calculations reveal a highly ordered, compact conformation in which a helical region extending from K9 to C15 lies in close apposition with the C-terminal hexapeptide; this interaction seems to be largely driven by hydrophobic interactions. Structure-activity studies are interpreted in terms of the conformational features of the calculated endothelin-3 structure.     

Study of the spatial structure of des-Gly9-[Arg8]vasopressin by two-dimensional NMR spectroscopy and theoretical conformation analysis

2D 1H-NMR spectra of des-Gly9-[Arg8]vasopressin in dimethylsulfoxide have been taken and the 1H resonances have been assigned. The coupling constants and amide proton temperature coefficients (delta delta/delta T) have been measured and the NOE cross-peaks in the NOESY spectrum have been analyzed. The most essential information on the spatial structure of des-Gly9-[Arg8]vasopressin is extracted from the low delta delta/delta T value for Asn5 amide proton and from the NOE between the Cys1 and Cys6 alpha-protons. A diminished accessibility of the Asn5 NH proton for the solvent is ascribed to the presence of a beta-turn in the fragment 2-5. The distance between the Cys1 and Cys6 C alpha H protons seems to be less than 4 A. These constraints were taken into account in the conformational analysis of the title peptide. The derived set of the low-energy backbone conformations was analyzed against the background of the all available NMR data. The most probable conformation of the cyclic moiety in des-Gly9-[Arg8]vasopressin was found to be the type III beta-turn. The corner positions are occupied by the residues 3, 4, while the residues 1-2 and 5-6 are at the extended sites. Some NMR data indicate that this structure is in a dynamic equilibrium with other minor conformers.     

A 30-residue fragment of the carp granulin-1 protein folds into a stack of two beta-hairpins similar to that found in the native protein.

Upon air oxidation, a peptide corresponding to the 30-residue N-terminal subdomain of carp granulin-1 spontaneously formed the disulfide pairing observed in the native protein. Structural characterization using NMR showed the presence of a defined secondary structure within this peptide. The chemical shifts for most of the alphaCH protons of the peptide and the protein are very similar, and the observed NOE contacts of the peptide strongly resemble those in the protein. A structure calculation of the peptide using NOE distance constraints indicates that the peptide fragment adopts the same conformation as formed within the native protein. The 30-residue N-terminal peptide of carp granulin-1 is the first example of an independently folded stack of two beta-hairpins reinforced by two interhairpin disulfide bonds. Two key areas of the structure show a clustering of hydrophobic residues that may account for its exceptional conformational stability.     

Conformation in solution of porcine brain natriuretic peptide determined by combined use of nuclear magnetic resonance and distance geometry

The conformation in solution of porcine brain natriuretic peptide was determined by combined use of NMR spectroscopy and distance geometry. A set of 157 inter-proton-distance constraints was derived from the two-dimensional NOE spectra, and further a set of three hydrogen bond constraints was obtained from analysis of the temperature dependence of labile protons. The five structures with minimal violations were selected after performing distance-geometry calculations starting from 40 random initial conformations. The distance-geometry structures were further refined by the use of restrained energy minimization and restrained molecular dynamics. This structure shows a compact conformation with the carboxy-terminal region, Asn21-Tyr26, folded back to the disulfide-linked loop region, Cys4-Cys20. The characteristics of the conformation determined are as follows: conformations of the three segments interposed by glycine residues, which are Arg7-Ile12, Ser14-Leu18 and Cys20-Arg25, were well defined and the segments Arg7-Ile12 and Cys20-Arg25 are rather close to each other and nearly parallel. The biological significance of these local conformations is discussed on the basis of comparisons with those of atrial natriuretic peptide reported by Kobayashi et al.     

Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH.

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.     

The conformational effects of N-glycosylation on the tailpiece from serum IgM

1H-NMR spectroscopy has been used to study the conformation and dynamics of the isolated tailpiece from human serum immunoglobulin M, a 22-residue peptide containing a single asparagine glycosylation site. The peptide is isolated as a set of glycoforms, varying only in the sequence of the oligosaccharide attached at the glycosylation site. The oligosaccharides present have the general formula (Man)n(GlcNAc)2, with 45% having n = 6, 45% having n = 8 and 10% having n = 7 and/or 9. They have been identified and their NMR parameters compared to those found for the isolated oligosaccharides in free solution. The conformation and dynamics of the peptide component have also been studied, using NOE data and hydrogen-exchange experiments, and the results compared to those obtained from the aglycosyl peptide of the same sequence. The presence of the peptide is found to have no measurable effect on the conformation of the oligosaccharides. However, the presence of oligosaccharide causes a decrease in the conformational mobility of the backbone and sidechains of the peptide in the region of the glycosylation site. This is proposed to result from interactions between the oligosaccharide core and the amino acid side chains. Further, the conformation of the N-glycosidic linkage has been shown to be both rigid and planar. Thus, the conformational space available to an N-linked oligosaccharide in a glycoprotein relative to the protein may depend to a large extent upon the flexibility of the asparagine side chain. Various roles for the different glycoforms of the tail peptide are discussed.     

The effect of pH on colchicine conformation and structure.

The effect of various pH values between 0 and 14 on the structure and conformation of colchicine was examined using UV-vis spectrophotometry at a concentration of 1.7 x 10(-5) M and NMR techniques at a colchicine concentration of 0.1M. The complete interpretation of the colchicine NMR spectrum in D2O is given. A stable structure of the colchicine molecule in aqueous solutions at pH from 2 to 12 was demonstrated. However, during incubation at 40 degrees C colchicine was found to be stable only at pH values between 2 and 10. The significance of these data for reactions of cholchicine in regard to metabolism and interaction with macromolecules is discussed.     

Solution structures of human transforming growth factor alpha derived from 1H NMR data

The 600-MHz 1H NMR spectrum of the des-Val-Val mutant of human transforming growth factor alpha (TGF-alpha) was reassigned at pH = 6.3. The conformation space of des-Val-Val TGF-alpha was explored by distance geometry embedding followed by restrained molecular dynamics refinement using NOE distance constraints and some torsion angle constraints derived from J-couplings. Over 80 long-range NOE constraints were found by completely assigning all resolved cross-peaks in the NOESY spectra. Low NOE constraint violations were observed in structures obtained with the following three different refinement procedures: interactive annealing in DSPACE, AMBER 3.0 restrained molecular dynamics, and dynamic simulated annealing in XPLOR. The segment from Phe15 to Asp47 was found to be conformationally well-defined. Back-calculations of NOESY spectra were used to evaluate the quality of the structures. Our calculated structures resemble the ribbon diagram presentations that were recently reported by other groups. Several side-chain conformations appear to be well-defined as does the relative orientation of the C loop to the N-terminal half of the protein.     

Non-Watson-Crick structures in oligodeoxynucleotides: self-association of d(TpCpGpA) stabilized at acidic pH

The 1H NMR spectrum of the tetradeoxynucleotide d(TpCpGpA) was examined as a function of temperature, pH, and concentration. At pH 7 and above the solution conformation for this oligodeoxynucleotide appears to be a mixture of random coil and Watson-Crick duplex. At 25 degrees C, a pH titration of d(TpCpGpA) shows that distinct conformational changes occur as the pH is lowered below 7.0. These conformational changes are reversible upon readjusting the pH to neutrality, indicating the presence of a pH-dependent set of conformational equilibria. At 25 degrees C, the various conformational states in the mixture are in rapid exchange on the NMR time scale. Examination of the titration curve shows the presence of distinct conformational states at pH greater than 7, and between pH 4 and pH 5. At pH less than 4, a third conformational state is present. When the pH titration is repeated at 5 degrees C, the conformational equilibria are in slow exchange on the NMR time scale; distinct signals from each conformational state are observable. The stable conformational state present between pH 4 and pH 5 represents an ordered conformation of d(TpCpGpA) which dissociates to a less ordered structure upon raising the temperature. This ordered conformation does not result from an intramolecular rearrangement, as is shown by by spectra obtained by varying oligodeoxynucleotide concentration at constant pH. The ordered conformation differs from the Watson-Crick helix, as is shown from nuclear Overhauser enhancement experiments, as well as chemical shift data. An ordered conformation for d(TpCpGpA) was previously reported [Reid, D. G., Salisbury, S. A., Brown, T., & Williams, D. H. (1985) Biochemistry 24, 4325-4332].(ABSTRACT TRUNCATED AT 250 WORDS)     

How to lose a kink and gain a helix: pH independent conformational changes of the fusion domains from influenza hemagglutinin in heterogeneous lipid bilayers

We have simulated two conformations of the fusion domain of influenza hemagglutinin (HA) within explicit water, salt, and heterogeneous lipid bilayers composed of POPC:POPG (4:1). Each conformation has seven different starting points in which the initial peptide structure is the same for each conformation, but the location across the membrane normal and lipid arrangement around the peptide are varied, giving a combined total simulation time of 140 ns. For the HA5 conformation (primary structure from recent NMR spectroscopy at pH = 5), the peptide exhibits a stable and less kinked structure in the lipid bilayer compared to that from the NMR studies. The relative fusogenic behavior of the different conformations has been investigated by calculation of the relative free energy of insertion into the hydrophobic region of lipid bilayer as a function of the depth of immersion. For the HA7 conformations (primary structure from recent NMR spectroscopy at pH = 7.4), while the N-terminal helix preserves its initial structure, the flexible C-terminal chain produces a transient helical motif inside the lipid bilayer. This conformational change is pH-independent, and is closely related to the peptide insertion into the lipid bilayer.     

-NH-dansyl isocolchicine exhibits a significantly improved tubulin-binding affinity and microtubule inhibition in comparison to isocolchicine by binding tubulin through its A and B rings

Structure-activity relationship studies have established that the A and C rings of colchicine comprise the minimum structural feature necessary for high affinity drug-tubulin binding. Thus, colchicine acts as a bifunctional ligand by making two points of attachment to the protein. Furthermore, analogues belonging to the iso series of colchicine are virtually inactive in binding to tubulin and inhibiting microtubule assembly. In the present study, we found that the substitution of a hydrophobic dansyl group on the B-ring side chain (C7 position) of isocolchicine reverses the structural alterations at the C ring and the newly synthesized -NH-dansyl isocolchicine restores the lost biological activity of the compound. It inhibits microtubule assembly efficiently with an IC(50) value of 10 microM and competes with [(3)H]colchicine for binding to tubulin. Moreover, although -NH-dansyl colchicine binding to tubulin involves two steps, the -NH-dansyl isocolchicine-tubulin interaction has been found to occur via a one-step process. Also, the affinity constant of the -NH-dansyl isocolchicine-tubulin interaction is roughly only 3 times lower than that of the -NH-dansyl colchicine-tubulin interaction. These results suggest that the enhanced microtubule inhibitory ability of -NH-dansyl isocolchicine is therefore related to the affinity of the drug-tubulin interaction and not to any conformational changes upon binding tubulin. We also observed that the competition of -NH-dansyl isocolchicine with [(3)H]colchicine for binding to tubulin was dependent on the tubulin concentration. In conclusion, this paper for the first time indicates that a biologically active bifuntional colchicine analogue can be designed where the drug binds tubulin through its A and B rings, while the C ring remains inactive.     

NMR studies of amyloid beta-peptides: proton assignments, secondary structure, and mechanism of an alpha-helix----beta-sheet conversion for a homologous, 28-residue, N-terminal fragment.

Beta-peptide is a major component of amyloid deposits in Alzheimer's disease. We report here a proton nuclear magnetic resonance (NMR) spectroscopic investigation of a synthetic peptide that is homologous to residues 1-28 of beta-peptide [abbreviated as beta-(1-28)]. The beta-(1-28) peptide produces insoluble beta-pleated sheet structures in vitro, similar to the beta-pleated sheet structures of beta-peptide in amyloid deposits in vivo. For peptide solutions in the millimolar range, in aqueous solution at pH 1-4 the beta-(1-28) peptide adopts a monomeric random coil structure, and at pH 4-7 the peptide rapidly precipitates from solution as an oligomeric beta-sheet structure, analogous to amyloid deposition in vivo. The NMR work shown here demonstrates that the beta-(1-28) peptide can adopt a monomeric alpha-helical conformation in aqueous trifluoroethanol solution at pH 1-4. Assignment of the complete proton NMR spectrum and the determination of the secondary structure were arrived at from interpretation of two-dimensional (2D) NMR data, primarily (1) nuclear Overhauser enhancement (NOE), (2) vicinal coupling constants between the amide (NH) and alpha H protons, and (3) temperature coefficients of the NH chemical shifts. The results show that at pH 1.0 and 10 degrees C the beta-(1-28) peptide adopts an alpha-helical structure that spans the entire primary sequence. With increasing temperature and pH, the alpha-helix unfolds to produce two alpha-helical segments from Ala2 to Asp7 and Tyr10 to Asn27. Further increases in temperature to 35 degrees C cause the Ala2-Asp7 section to become random coil, while the His13-Phe20 section stays alpha-helical. A mechanism involving unfavorable interactions between charged groups and the alpha-helix macrodipole is proposed for the alpha-helix----beta-sheet conversion observed at midrange pH.     

A helix-turn motif in the C-terminal domain of histone H1

The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs.