Nuclear Overhauser effect and computational characterization of the beta-spiral of the polypentapeptide of elastin

The structure of the elastin polypentapeptide, poly(VPGVG), was studied by nuclear Overhauser effect experiments using perdeuterated Val1 and Val4 samples under the condition where intermolecular interactions are absent. More extensive interaction was found between the Val1 gamma CH and Pro2 beta CH protons than between the Val4 gamma CH and Pro2 beta CH protons. The Val1 gamma CH3-Pro2 beta CH interaction does not occur within the same pentamer as previously shown experimentally and as expected from steric considerations. The results are incompatible with the presence of a random chain network in poly(VPGVG) at room temperature but are readily explicable in terms of interturn interactions in a beta-spiral structure. More specifically, the results indicate that the beta-spiral conformation with 2.9 pentamers/turn is more prevalent than that with 2.7 pentamers/turn. Using conformations developed by molecular mechanics calculations, molecular dynamics simulations were carried out to compare the relative energies of these two variants of this class of beta-spiral structures. It was found in vacuo that the structure with 2.9 pentamers/turn is indeed more stable than that of 2.7 pentamers/turn by approximately 1 kcal/mole-pentamer.     

Solution conformations of two flexible cyclic pentapeptides: cyclo(Gly-Pro-D-Phe-Gly-Ala) and cyclo(Gly-Pro-D-Phe-Gly-Val).

In an effort to explore the residue preferences in three-residue reverse turns (so-called gamma-turns), two cyclic pentapeptides--cyclo(Gly1-Pro2-D-Phe3-Gly4-Ala5) (I) and cyclo(Gly1-Pro2-D-Phe3-Gly4-Val5) (II)--have been synthesized and analyzed by nmr. It was anticipated that the Gly-Pro-D-Phe-Gly portions of these molecules would favor a beta-turn conformation, leaving the remainder of the molecule to adopt a gamma turn, as seen in several previously studied model cyclic pentapeptides. The nmr data for both peptides in CDCl3 (5% DMSO-d6) and in neat DMSO-d6 indicate that the most populated conformation contains a distorted beta turn around Pro2-D-Phe3, which includes a gamma turn around D-Phe3. The distortion in the beta turn does not impede the formation of an inverse gamma turn around residue 5, and indeed, this conformation is observed in both peptides. Both the alanine and the bulkier valine residues are therefore found to be compatible with an inverse gamma turn. Molecular dynamics simulations on the title peptides are reported in the following paper. These simulations indicate that there is conformational flexibility around the D-Phe3-Gly4 peptide bond, which enables the formation of the gamma turn around D-Phe3. The third paper in this series explores the impact of a micellar environment on conformational equilibria in II.     

Polytetrapeptide of elastin. Temperature-correlated elastomeric force and structure development

High molecular weight polytetrapeptide of elastin, (L.Val1-L.Pro2-Gly3-Gly4)n, was synthesized using activation of the (GGVP) permutation for polymerization. The temperature-dependence of aggregation was characterized as a function of concentration and the circular dichroism spectra were obtained in the 20 degrees to 70 degrees C temperature range. The latter showed an inverse temperature transition centered near 50 degrees C in which polypeptide order increased on raising the temperature. A concentration of 0.6 g of polytetrapeptide in 1 g of water was gamma irradiation cross-linked (20 Mrad) to form an elastomeric matrix. A study of the temperature-dependence of elastomeric force demonstrated a transition toward increased force on raising the temperature with a midpoint of the transition near 50 degrees C. Thus, there is a correlation between increase in intramolecular order and elastomeric force development. These results are compared to previous results on the polypentapeptide of elastin, (VPGVG)n and on an analog, (IPGVG)n, to demonstrate that the temperature of the transition is proportional to the hydrophobicity of the repeating unit. The point is noted that the elastomeric force development correlates better with intramolecular ordering than with intermolecular processes.     

Conformations of an ion-binding cyclic peptide analogue of valinomycin, cyclo(L-Val-Gly-Gly-L-Pro)3.

A 270-MHz 1H nuclear magnetic resonance investigation of an ion-binding cyclic peptide analogue of valinomycin, cyclo(L-Val-Gly-Gly-L-Pro)3, and its cation complexes is reported. In CD2Cl2 and CDCl3, the peptide is proposed to occur in a C3-symmetric conformer with the N--H's of all six glycine residues intramolecularly hydrogen bonded. This conformation is different from the familiar valinomycin bracelet structure and lacks any "cavity". Cations do not bind, or bind only weakly, to the peptide in these solvents. Uncomplexed cyclo(L-Val-Gly-Gly-L-Pro)3 in acetonitrile appears to be averaging among several conformations with no evidence found for any preferred intramolecular hydrogen bonds. The strong 1:1 complexes of cyclo(L-Val-Gly-Gly-L-Pro)3 with K+ ANd Ba2+ in acetonitrile are structurally analogous to the bracelet conformation of valinomycin and involve the N--H's of the Val residues and of the Gly's preceding Pro in intramolecular hydrogen bonding. Tl+ was also found to form strong 1:1 complexes with the dodecapeptide.     

Structural requirements essential for elastin coacervation: favorable spatial arrangements of valine ridges on the three‐dimensional structure of elastin‐derived polypeptide (VPGVG)n

The elastin precursor tropoelastin possesses a number of polymeric peptides with repeating 3–9 mer sequences. One of these is the pentapeptide Val‐Pro‐Gly‐Val‐Gly (VPGVG) present in almost all animal species, and its polymer (VPGVG)n coacervates just as does tropoelastin. In the present study, in order to explore the structural requirements essential for coacervation, (VPGVG)n and its shortened repeat analogs (VPGV)n, (VPG)n, and (PGVG)n were synthesized and their structural properties were investigated. In our turbidity measurements, (VPGVG)n demonstrated complete reversible coacervation in agreement with previous findings. The Gly⁵‐deleted polymer (VPGV)n also achieved self‐association, though the onset of self‐association occurred at a lower temperature. However, the dissociation of (VPGV)n upon temperature lowering was found to occur in a three‐step process; the Val_i⁴‐Val_i+1¹ structure arising in the VPGV polypeptide appeared to perturb the dissociation. No self‐association was observed for (VPG)n or (PGVG)n repeats. Spectroscopic measurements by CD, FT‐IR, and ¹H‐NMR showed that the (VPGV)n and (VPG)n both assumed ordered structures similar to that of (VPGVG)n. These results demonstrated that VPGVG is a structural element essential to achieving the β‐spiral structure required for self‐association followed by coacervation, probably due to the ideal spatial arrangement of the hydrophobic Val residues. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Combined use of molecular dynamics simulations and NMR to explore peptide bond isomerization and multiple intramolecular hydrogen-bonding possibilities in a cyclic pentapeptide, cyclo(Gly-Pro-D-Phe-Gly-Val).

The conformational behavior of a model cyclic pentapeptide--cyclo(Gly-L-Pro-D-Phe-Gly-L-Val)--has been explored through the combined use of in vacuo molecular dynamics simulations and a range of nmr experiments (preceding paper). The molecular dynamics analysis suggests that, despite the conformational constraints imposed by formation of the pentapeptide cycle, this pentapeptide undergoes conformational transitions between various hydrogen-bonded conformations, characterized by low energy barriers. An inverse gamma turn with Pro in position i + 1 and a gamma turn with D-Phe in position i + 1 are two alternatives occurring frequently. Like other DLDDL cyclic pentapeptides, cyclo(Gly-Pro-D-Phe-Gly-Val) is also stabilized by an inverse gamma-turn structure with the beta-branched Val residue in position i + 1, and this hydrogen bond is retained in the different conformational families. The gamma-turn around D-Phe3 and the inverse gamma turn around Val5 are consistent with the nmr observations. 3JNH-CH alpha coupling constants of the all-trans forms were calculated from one of the molecular dynamics trajectories and are comparable to nmr experimental data, suggesting that the conformational states visited during the simulation are representative of the conformational distribution in solution. In addition to the equilibrium among various hydrogen-bonded all-trans conformers, the observation in nmr spectra of two sets of resonances for all peptide protons indicated a slow conformational interconversion of the Gly-Pro peptide bond between trans and cis isomers. The activation energy between these two conformers was determined experimentally by magnetization transfer and was calculated by high temperature constrained molecular dynamics simulation. Both methods yield a free energy of activation of ca. 20 kcal/mol. Furthermore, the free energy of activation is dependent on the direction of rotation of the Gly-Pro peptide bond.     

Temperature-induced conformational transition of a model elastin-like peptide GVG(VPGVG)(3) in water

The conformation of a single elastin-like peptide GVG(VPGVG)3 in liquid water is studied by computer simulations in the temperature interval between 280 and 440 K. Two main conformational states of the peptide can be distinguished: a rigid conformational state, dominating at low temperatures, and a flexible conformational state, dominating at high temperatures. A temperature-induced transition between these states occurs at about 310 K, rather close to a transition temperature seen in experiments. This transition is accompanied by the thermal breaking of the hydrogen-bonded spanning network of the hydration water via a percolation transition upon heating. This finding indicates that the H-bond clustering structure of the hydration water plays an important role in the conformational stability of biomolecules. A second important observation is the Gaussian distribution of the end-to-end distance in the high-temperature state, which supports the idea of a rubber-like elasticity of the studied elastin-like peptide. Finally our results challenge the idea of the folding of elastin-like peptides upon heating.     

Crystal structure of (Gly-Pro-Hyp)(9) : implications for the collagen molecular model

Collagens have long been believed to adopt a triple‐stranded molecular structure with a 10/3 symmetry (ten triplet units in three turns) and an axial repeat of 29 Å. This belief even persisted after an alternative structure with a 7/2 symmetry (seven triplet units in two turns) with an axial repeat of 20 Å had been proposed. The uncertainty regarding the helical symmetry of collagens is attributed to inadequate X‐ray fiber diffraction data. Therefore, for better understanding of the collagen helix, single‐crystal analyses of peptides with simplified characteristic amino acid sequences and similar compositions to collagens have long been awaited. Here we report the crystal structure of (Gly‐Pro‐Hyp)₉ peptide at a resolution of 1.45 Å. The repeating unit of this peptide, Gly‐Pro‐Hyp, is the most typical sequence present in collagens, and it has been used as a basic repeating unit in fiber diffraction analyses of collagen. The (Gly‐Pro‐Hyp)₉ peptide adopts a triple‐stranded structure with an average helical symmetry close to the ideal 7/2 helical model for collagen. This observation strongly suggests that the average molecular structure of collagen is not the accepted Rich and Crick 10/3 helical model but is a 7/2 helical conformation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 607–616, 2012.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

Production and purification of a recombinant elastomeric polypeptide, G-(VPGVG)19-VPGV, from Escherichia coli.

An elastomeric polypeptide was produced, with the sequence G-(VPGVG)19-VPGV, as a fusion to glutathione S-transferase using the vector pGEX-3X. The fusion protein was expressed to high levels in Escherichia coli as indicated by SDS-PAGE analysis of induced cells. The fusion protein was affinity purified and cleaved with protease factor Xa, and the elastomeric polypeptide was recovered to a high degree of purity as indicated by SDS-PAGE followed by staining with CuCl2. The physical characterizations of carbon-13 and proton nuclear magnetic resonance and of the temperature profile for turbidity formation for the inverse temperature transition of hydrophobic folding and assembly attest to the successful microbial synthesis of the polypentapeptide of elastin. The results of these studies provide the initial progress toward achieving a more economical and practical means of producing material for elastic protein-based polymer research and applications.     

Designing of peptides with left handed helical structure by incorporating the unusual amino acids

The conformational behaviour of delta Ala has been investigated by quantum mechanical method PCILO in the model dipeptide Ac-delta Ala-NHMe and in the model tripeptides Ac-X-delta Ala-NHMe with X = Gly, Ala, Val, Leu, Abu and Phe and is found to be quite different. The computational results suggest that in the model tripeptides the most stable conformation corresponds to phi 1 = -30 degrees, psi 1 = 120 degrees and phi 2 = psi 2 = 30 degrees in which the > C = 0 of the acetyl group is involved in hydrogen bond formation with N-H of the amide group. Similar results were obtained for the conformational behaviour of D-Ala in Ac-D-Ala-NHMe and Ac-Ala-D-Ala-NHMe. The conformational behaviour of the amino acids delta Ala, D-Ala, Val and Aib in model tripeptides have been utilized in the designing of left handed helical peptides. It is shown that the peptide HCO-(Ala-D-Ala)3-NHMe can adopt both left and right handed helix whereas in the peptide Ac-(Ala-delta Ala)3-NHMe the lowest energy conformer is beta-bend ribbon structure. Left handed helical structure with phi = 30 degrees, psi = 60 degrees for D-Ala residues and phi = psi = 30 degrees for delta Ala is found to be more stable by 4 kcal mole-1 than the corresponding right handed helical structure for the peptide Ac-(D-Ala-delta Ala)3-NHMe. In both the peptides Ac-(Val-delta Ala)3-NHMe and Ac-(D-Val-delta Ala)3-NHMe the most stable conformer is the left handed helix. Comparisons of results for Ac-(Ala-delta Ala)3-NHMe and Ac(Val-delta Ala)3-NHMe and Ac-(D-Ala-delta Ala)3-NHMe and Ac-(D-Val-delta Ala)3-NHMe also reveal that the Val residues facilitate the population of 3(10) left handed helix over the other conformers. It is also shown that the conformational behaviour of Aib residue depends on the chirality of neighbouring amino acids, i.e. Ac-(Aib-Ala)3-NHMe adopts right handed helical structure whereas Ac-(Aib-D-Ala)3-NHMe is found to be in left handed helical structure.     

Structure of an elastin-mimetic polypeptide by solid-state NMR chemical shift analysis

The conformation of an elastin-mimetic recombinant protein, [(VPGVG)4(VPGKG)]39, is investigated using solid-state NMR spectroscopy. The protein is extensively labeled with 13C and 15N, and two-dimensional 13C-13C and 15N-13C correlation experiments were carried out to resolve and assign the isotropic chemical shifts of the various sites. The Pro 15N, 13Calpha, and 13Cbeta isotropic shifts, and the Gly-3 Calpha isotropic and anisotropic chemical shifts support the predominance of type-II beta-turn structure at the Pro-Gly pair but reject a type-I beta-turn. The Val-1 preceding Pro adopts mostly beta-sheet torsion angles, while the Val-4 chemical shifts are intermediate between those of helix and sheet. The protein exhibits a significant conformational distribution, shown by the broad line widths of the 15N and 13C spectra. The average chemical shifts of the solid protein are similar to the values in solution, suggesting that the low-hydration polypeptide maintains the same conformation as in solution. The ability to measure these conformational restraints by solid-state NMR opens the possibility of determining the detailed structure of this class of fibrous proteins through torsion angles and distances.     

Conformational Analysis of a Hybrid DNA-RNA Double Helical Oligonucleotide in Aqueous Solution: d(CG)r(CG)d(CG) Studied by 1D- and 2D-1H NMR Spectroscopy

Abstract

The double helical structure of the self-complementary DNA-RNA-DNA hybrid d(CG)r(CG) d(CG) was studied in solution by 500 MHz ¹H-NMR spectroscopy. The non-exchangeable base protons and the (deoxy)ribose H1′, H2′ and H2″ protons were unambiguously assigned using 2D-J-correlated (COSY) and 2D-NOE (NOESY) spectroscopy techniques. A general strategy for the sequential assignment of ¹H-NMR spectra of (double) helical DNA and RNA fragments by means of 2D-NMR methods is presented.

Conformational analysis of the sugar rings of d(CG)r(CG)d(CG) at 300 K shows that the central ribonucleotide part of the helix adopts an A-type double helical conformation. The 5′- and 3′-terminal deoxyribose base pairs, however, take up the normal DNA-type conformation. The A-to-B transition in this molecule involves only one (deoxyribose) base pair. It is shown that this A-to-B conformational transition can only be accomodated by two specific sugar pucker combinations for the junction base pair, i.e. N·S (C3′-endo-C2′-endo, 60%, where the pucker given first is that assigned to the junction nucleotide residue of the strand running 5′ → 3′ from A-RNA to B-DNA) and S·S (C2′-endo-C2′-endo, 40%).     

Spectroscopic characterization of poly(Glu-Ala)

Infrared linear dichroism and ultraviolet circular dichroism speetroscopy have been used to distinguish four conformational forms of the ionizable sequential polypeptide poly(Glu-Ala). Two of these conformations, the α helix and the β form, were observed for the unionized polypeptide in solution. The α helix appeared immediately upon neutralization of the side-chain carboxyl functions, whereas the β form was observed after the neutralized solution had been standing for several days. The β form was also observed for films cast from either high or low pH solutions. Ionization of the glutamyl residues resulted in a circular dichroism spectrum which has previously been observed for charged homopolymers and appears to result from an extended helical conformation. Further, heating either the α helical or the charged extended helix resulted in a transition to a disordered chain. These results are consistent with the results of conformational calculations presented elsewhere.     

Effect of the N2 residue on the stability of the α-helix for all 20 amino acids

N2 is the second position in the alpha-helix. All 20 amino acids were placed in the N2 position of a synthetic helical peptide (CH(3)CO-[AXAAAAKAAAAKAAGY]-NH(2)) and the helix content was measured by circular dichroism spectroscopy at 273K. The dependence of peptide helicity on N2 residue identity has been used to determine a free-energy scale by analysis with a modified Lifson-Roig helix-coil theory that includes a parameter for the N2 energy (n2). The rank order of DeltaDeltaG((relative to Ala)) is Glu(-), Asp(-) > Ala > Glu(0), Leu, Val, Gln, Thr, Ile, Ser, Met, Asp(0), His(0), Arg, Cys, Lys, Phe > Asn, > Gly, His(+), Pro, Tyr. The results correlate very well with N2 propensities in proteins, moderately well with N1 and helix interior preferences, and not at all with N-cap preferences. The strongest energetic effects result from interactions with the helix dipole, which favors negative charges at the helix N terminus. Hydrogen bonds to side chains at N2, such as Gln, Ser, and Thr, are weak, despite occurring frequently in protein crystal structures, in contrast to the N-cap position. This is because N-cap hydrogen bonds are close to linear, whereas N2 hydrogen bonds have poor geometry. These results can be used to modify protein stability rationally, help design helices, and improve prediction of helix location and stability.     

Nuclear Overhauser Effect and Computational Characterization of the β-Spiral of the Polypentapeptide of Elastin

Abstract

The structure of the elastin polypentapeptide, poly(VPGVG), was studied by nuclear Overhauser effect experiments using perdeuterated Val¹ and Val⁴ samples under the condition where intermolecular interactions are absent. More extensive interaction was found between the Val¹ γCH and Pro² βCH protons than between the Val⁴ γCH and Pro² βCH protons. The Val¹ γCH₃—Pro² βCH interaction does not occur within the same pentamer as previously shown experimentally and as expected from steric considerations. The results are incompatible with the presence of a random chain network in poly(VPGVG) at room temperature but are readily explicable in terms of interturn interactions in a β-spiral structure. More specifically, the results indicate that the β-spiral conformation with 2.9 pentamers/turn is more prevalent than that with 2.7 pentamers/turn. Using conformations developed by molecular mechanics calculations, molecular dynamics simulations were carried out to compare the relative energies of these two variants of this class of β-spiral structures. It was found in vacuo that the structure with 2.9 pentamers/turn is indeed more stable than that of 2.7 pentamers/turn by ~ 1 kcal/mole-pentamer.     

Determination of interproton distances in peptides by two-dimensional nuclear Overhauser effect spectroscopy (NOESY). Conformation of a cyclic analog of substance P in a solution

Quantitative method is developed for evaluation interproton distances in peptides in solution. The method is based on the measurement of the relative intensities of the cross-peaks in the pure-phase absorption NOESY spectra. The ratios of the cross-peak intensities IN alpha/I alpha N and INN/I alpha N enable to determine the corresponding interproton distances dN alpha, d alpha N and dNN for several amino acid residues. These distances can be used to estimate other distances with cross-peaks in NOESY spectra. As example, the interproton distances are determined in a cyclic hexapeptide, namely cyclic analogue of substance P: cyclo [H-Glu-Phe-Phe-Gly-Leu-Met-NH(CH2)3-NH-]. The spatial structure of the molecule in dimethylsulphoxide solution is established.     

Tuning the beta-turn segment in designed peptide beta-hairpins: construction of a stable type I' beta-turn nucleus and hairpin-helix transition promoting segments

Designed octapeptides Boc-Leu-Val-Val-Aib-(D)Xxx-Leu-Val-Val-OMe ((D)Xxx = (D)Ala, 3a;(D)Val, 3c and (D)Pro, 5a) and Boc-Leu-Phe-Val-Aib-(D)Ala-Leu-Phe-Val-OMe (3b) have been investigated to construct models of a stable type I' beta-turn nucleated hairpin and to generate systems for investigating helix-hairpin conformational transitions. Peptide 5a, which contains a central Aib-(D)Pro segment, is shown to adopt a stable type I' beta-turn nucleated hairpin structure, stabilized by four cross-strand hydrogen bonds. The stability of the structure in diverse solvents is established by the observation of all diagnostic NOEs expected in a beta-hairpin conformation. Replacement of (D)Pro5 by (D)Ala/(D)Val (3a-c) results in sequences that form beta-hairpins in hydrogen bonding solvents like CD(3)OH and DMSO-d(6). However, in CDCl(3) evidence for population of helical conformations is obtained. Peptide 6b (Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe), which contains a centrally positioned Aib-Aib segment, provides a clear example of a system, which exhibits a helical conformation in CDCl(3) and a significant population of both helices and hairpins in CD(3)OH and DMSO-d(6). The coexistence of multiple conformations is established by the simultaneous observation of diagnostic NOEs. Control over stereochemistry of the central beta-turn permits generation of models for robust beta-hairpins and also for the construction of systems that may be used to probe helix-hairpin conformational transitions.     

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.