Helix-sheet interconversion in a synthetic pore lining peptide derived from a designed ion channel

We have designed a four-helix protein that is expected to tetramerize in the membrane to form an ion channel with a structurally well defined pore. A synthetic peptide corresponding to the channel lining helix facilitates ion transport across liposomal membranes and largely helical in membranes. Detailed circular dichroism studies of the peptide in methanol, water and methanal-water mixtures reveal that it is helical in methanol, beta-structured in 97.5% water and a combination of these two structures at intermediate compositions of methanol and water. A fluorescence resonance energy transfer study of the peptide shows that the peptide is monomeric in methanol but undergoes extensive anti-parallel aggregation in aqueous solution.

The crystal structure of the amino-terminal pentapeptide of suzukacillin. Occurrence of a four-fold peptide helix

The monohydrate of the protected amino-terminal pentapeptide of suzukacillin, t-butoxycarbonyl--aminoisobutyryl-L-prolyl-L-valyl--aminoisobutyryl-L-valine methyl ester, C29H51N5O8, crystallizes in the orthorhombic space group P212121 with a= 10.192, b= 10.440, c= 32.959 Å, and Z= 4. The structure has been solved by direct methods and refined to an R value of 0.101 for 1 827 observed reflections. The molecule exists as a four-fold helix with a pitch of 5.58 Å. The helix is stabilised by N–H O hydrogen bonds, two of the 51 type (corresponding to the -helix) and the third of the 41 type (310 helix). The carbonyl oxygen of the amino-protecting group accepts two hydrogen bonds, one each from the amide NH groups of the third (41) and fourth (51) residues. The remaining 51 hydrogen bond is between the two terminal residues. The lone water molecule in the structure is hydrogen bonded to carbonyl oxygens of the prolyl residue in one molecule and the non-terminal valyl residue in a symmetry-related molecule.

The poly dA helix: a new structural motif for high performance DNA-based molecular switches

We report a pH-dependent conformational transition in short, defined homopolymeric deoxyadenosines (dA(15)) from a single helical structure with stacked nucleobases at neutral pH to a double-helical, parallel-stranded duplex held together by AH-HA base pairs at acidic pH. Using native PAGE, 2D NMR, circular dichroism (CD) and fluorescence spectroscopy, we have characterized the two different pH dependent forms of dA(15). The pH-triggered transition between the two defined helical forms of dA(15) is characterized by CD and fluorescence. The kinetics of this conformational switch is found to occur on a millisecond time scale. This robust, highly reversible, pH-induced transition between the two well-defined structured states of dA(15)represents a new molecular building block for the construction of quick-response, pH-switchable architectures in structural DNA nanotechnology.

Helix-coil stability constants for amino acids in nonaqueous solvents. Studies of random poly(-benzyl-L-glutamate-co-L-alanine) and poly(-benzyl-L-glutamate-co-L-leucine) in a mixed solvent

The host-guest technique has been applied to the determination of the helix-coil stability constants of two naturally occurring amino acids, L-alanine and L-leucine, in a nonaqueous solvent system. Random copolymers containing L-alanine and L-leucine, respectively, as guest residues and -benzyl-L-glutamate as the host residue were synthesized. The polymers were fractionated and characterized for their amino acid content, molecular weight, and helix-coil transition behavior in a dichloroacetic acid (DCA)-1,2-dichloroethane (DCE) mixture. Two types of helix-coil transitions were carried out on the copolymers: solvent-induced transitions in DCA-DCE mixtures at 25°C and thermally induced transitions in a 82:18 (wt %) DCA-DCE mixture. The thermally induced transitions were analyzed by statistical mechanical methods to determine the Zimm-Bragg parameters, and s, of the guest residues. The experimental data indicate that, in the nonaqueous solvent, the L-alanine residue stabilizes the -helical conformation more than the L-leucine residue does. This is in contrast to their behavior in aqueous solution, where the reverse is true. The implications of this finding for the analysis of helical structures in globular proteins are discussed.

Cyclic Peptide Disulfides - Solution And Solid-State Conformation Of Boc-Cys-Pro-Aib-Cys-S-S-Bridge-Nhme,A Disulfide-Bridged Peptide Helix

The solution and solid-state conformations of the peptide disulfide Boc-Cys-Pro-Aib-Cys-NHMe have been determined by NMR spectroscopy and X-ray diffraction. The Cys(4) and methylamide NH groups are solvent shielded in CDCI3 and (CD,),SO, suggesting their involvement in intramolecular hydrogen bonding. On the basis of known stereochemical preferences of Pro and Aib residues, a consecutive @-turn structure is favored in solution. X-ray diffraction analysis reveals a highly folded 310 helical conformation for the peptide, with the S-S bridge lying approximately parallel to the helix axis, linking residues 1 and 4. The backbone conformational angles are Cys(1) 4 = -121.1', $ = 65.6"; Pro(2) 4 = -58.9', 4 = -34.0'; Aib(3) 4 = -61.8', $ = -17.9'; Cys(4) 4 = -70.5', $ = -18.6'. Two intramolecular hydrogen bonds are observed between Cys(1) CO--HN Cys(4) and Pro(2) CO--HNMe. The disulfide bond has a right-handed chirality, with a dihedral angle (xss) of 82'.

Sequence-dependent conformation of an A-DNA double helix: The crystal structure of the octamer d(G-G-T-A-T-A-C-C)

The crystal structures of the synthetic self-complementary octamer d(G-G-T-A-T-A-C-C) and its 5-bromouracil-containing analogue have been refined to R values of 20% and 14% at resolutions of 1·8 and 2·25 Å, respectively. The molecules adopt an A-DNA type double-helical conformation, which is minimally affected by crystal forces. A detailed analysis of the structure shows a considerable influence of the nucleotide sequence on the base-pair stacking patterns. In particular, the electrostatic stacking interactions between adjacent guanine and thymine bases produce symmetric bending of the double helix and a major-groove widening. The sugar-phosphate backbone appears to be only slightly affected by the base sequence. The local variations in the base-pair orientation are brought about by correlated adjustments in the backbone torsion angles and the glycosidic orientation. Sequence-dependent conformational variations of the type observed here may contribute to the specificity of certain protein-DNA interactions.

Self-Organization of n-Alkane Chains in Water: Length Dependent Crossover from Helix and Toroid to Molten Globule

We demonstrate a chain length dependent crossover in the structural properties of linear hydrocarbon (n-alkane) chains using detailed atomistic simulations in explicit water. We identify a number of exotic structures of the polymer chain through energy minimization of representative snapshots collected from molecular dynamics trajectory. While the collapsed state is ring-like (circular) for small chains (CnH2n+2; n <= 20) and spherical for very long ones (n = 100), we find the emergence of ordered helical structures at intermediate lengths (n similar to 40). We find different types of disordered helices and toroid-like structures at n = 60. We also report a sharp transition in the stability of the collapsed state as a function of the chain length through relevant free energy calculations. While the collapsed state is only marginally metastable for C20H42, a clear bistable free energy surface emerges only when the chain is about 30 monomers long. For n = 30, the polymer exhibits an intermittent oscillation between the collapsed and the coil structures, characteristic of two stable states separated by a small barrier.

A Case Study of the Conformation of Poly(alpha-aminoisobutyric acid): alpha- or 310-Helix

The relative stabilities of a- and Blo-helical structures for polymers of a-aminoisobutyric acid (Aib) have been worked out, using the classical potential energy functions. To make a comparative study, we have used Buckingham "6-exp" and Kitaigorodsky's potential functions. Conformational analysis of the dipeptide segment with Aib residue indicates the necessity for nonplanar distortion of the peptide unit, which is a common feature in the observed crystal structures with Aib residues. In the range of Aw -10 to +loo studied, a-helical conformations are preferred in the region -3" < Aw < +loo, and Blo-helical conformations are preferred in the region -3" > Aw > -10'. Minimum energy conformations for right-handed structures are found in the +ue region of Aw and correspondingly for left-handed structures in the -ue region of Aw. For Aw - 6", a-helical structures have four- or near fourfold symmetry with h - 1.5 A. Such a helix with n = 4 and h = 1.5 A is termed an a'-helix. This structure is found to be consistent with the electron diffraction data of Malcolm3 and energetically more favorable than the standard 310-helix.

An incipient 310 helix in Piv-Pro-Pro-Ala-NHMe as a model for peptide folding

The molecular mechanism of helix nucleation in peptides and proteins is not yet understood and the question of whether sharp turns in the polypeptide backbone serve as nuclei for protein folding has evoked controversy1,2. A recent study of the conformation of a tetrapeptide containing the stereochemically constrained residue alpha-aminoisobutyric acid, both in solution and the solid state, yielded a structure consisting of two consecutive beta-turns, leading to an incipient 310 helical conformation3,4. This led us to speculate that specific tri- and tetra-peptide sequences may indeed provide a helical twist to the amino-terminal segment of helical regions in proteins and provide a nucleation site for further propagation. The transformation from a 310 helical structure to an alpha-helix should be facile and requires only small changes in the phi and psi conformational angles and a rearrangement of the hydrogen bonding pattern5. If such a mechanism is involved then it should be possible to isolate an incipient 310 helical conformation in a tripeptide amide or tetrapeptide sequence, based purely on the driving force derived from short-range interactions. We have synthesised and studied the model peptide pivaloyl-Pro-Pro-Ala-NHMe (compound I) and provide here spectroscopic evidence for a 310 helical conformation in compound I.

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-DAla-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 CD3OH and DMSO-d(6). However, in CDCl3 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 CDCl3 and a significant population of both helices and hairpins in CD3OH 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. (c) 2006 Wiley Periodicals, Inc.

Disulfide conformation and design at helix N-termini

To understand structural and thermodynamic features of disulfides within an alpha-helix, a non-redundant dataset comprising of 5025 polypeptide chains containing 2311 disulfides was examined. Thirty-five examples were found of intrahelical disulfides involving a CXXC motif between the N-Cap and third helical positions. GLY and PRO were the most common amino acids at positions 1 and 2, respectively. The N-Cap residue for disulfide bonded CXXC motifs had average values of (-112 +/- 25.2 degrees, 106 +/- 25.4 degrees). To further explore conformational requirements for intrahelical disulfides, CYS pairs were introduced at positions N-Cap-3; 1,4; 7,10 in two helices of an Escherichia coli thioredoxin mutant lacking its active site disulfide (nSS Trx). In both helices, disulfides formed spontaneously during purification only at positions N-Cap-3. Mutant stabilities were characterized by chemical denaturation studies (in both oxidized and reduced states) and differential scanning calorimetry (oxidized state only). All oxidized as well as reduced mutants were destabilized relative to nSS Trx. All mutants were redox active, but showed decreased activity relative to wild-type thioredoxin. Such engineered disulfides can be used to probe helix start sites in proteins of unknown structure and to introduce redox activity into proteins. Conversely, a protein with CYS residues at positions N-Cap and 3 of an alpha-helix is likely to have redox activity.

Monoclinic polymorph of Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe(anhydrous). Parallel packing of 3(10)-/alpha-helices and a transition of helix type

The structures of two crystal forms of Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe have been determined. The triclinic form (P1, Z = 1) from DMSO/H2O crystallizes as a dihydrate (Karle, Sukumar & Balaram (1986) Proc, Natl, Acad. Sci. USA 83, 9284-9288). The monoclinic form (P2(1), Z = 2) crystallized from dioxane is anhydrous. The conformation of the peptide is essentially the same in both crystal system, but small changes in conformational angles are associated with a shift of the helix from a predominantly alpha-type to a predominantly 3(10)-type. The r.m.s. deviation of 33 atoms in the backbone and C beta positions of residues 2-8 is only 0.29 A between molecules in the two polymorphs. In both space groups, the helical molecules pack in a parallel fashion, rather than antiparallel. The only intermolecular hydrogen bonding is head-to-tail between helices. There are no lateral hydrogen bonds. In the P2(1) cell, a = 9.422(2) A, b = 36.392(11) A, c = 10.548(2) A, beta = 111.31(2) degrees and V = 3369.3 A for 2 molecules of C60H97N11O13 per cell.

Conformation of the flexible bent helix of Leu1-zervamicin in crystal C and a possible gating action for ion passage

The membrane channel-forming polypeptide, Leu(1)-zervamicin, Ac-Leu-Ile-Gln-Iva-Ile(5)-Thr-Aib-Leu-Aib-Hyp(10) -Gln-Aib-Hyp-Aib-Pro(15)-Phol (Aib: alpha-aminoisobutyric acid; Iva: isovaline; Hyp: 4-hydroxyproline; Phol: phenylalininol) has been analyzed by x-ray diffraction in a third crystal form. Although the bent helix is quite similar to the conformations found in crystals A and B, the amount of bending is more severe with a bending angle approximate to 47 degrees, The water channel formed by the convex polar faces of neighboring helices is larger at the mouth than in crystals A and B, and the water sites have become disordered. The channel is interrupted in the middle by a hydrogen bond between the OH of Hyp(10) and the NH2 of the Gln(11) of a neighboring molecule. The side chain of Gln(11) is wrapped around the helix backbone in an unusual fashion in order that it can augment the polar side of the helix. In the present crystal C there appears to be an additional conformation for the Gln(11) side chain (with approximate to 20% occupancy) that opens the channel for possible ion passage. Structure parameters for C85H140N18O22.xH(2)O.C2H5OH are space group P2(1)2(1)2(1), a = 10.337 (2) Angstrom, b = 28.387 (7) Angstrom, c = 39.864 (11) Angstrom, Z = 4, agreement factor R = 12.99% for 3250 data observed > 3 sigma(F), resolution = 1.2 Angstrom. (C) 1994 John Wiley & Sons, Inc.

Crystal structures of a nonapeptide helix containing alpha,alpha-di-n-butylglycine (Dbg), Boc-Gly-Dbg-Ala-Val-Ala-Leu-Aib-Val-Leu-OMe

Two crystals structures of a nonapeptide (anhydrous and hydrated) containing the amino acid residue alpha, alpha-di-n-butylglycyl, reveal a mixed 3(10)/alpha-helical conformation. Residues 1-7 adopt phi, psi values in the helical region, with Val(8) being appreciably distorted. The Dbg residue has phi, psi values of -40, -37 degrees and -46, -40 degrees in two crystals with the two butyl side chains mostly extended in each. Peptide molecules in the crystals pack into helical columns. The crystal parameters are C50H91N9O12, space group P2(1), with a = 9.789(1) Angstrom, b = 20.240(2) Angstrom, c = 15.998(3) Angstrom, beta = 103.27(1); Z = 2, R = 10.3% for 1945 data observed >3 sigma(F) and C50H91N9O12. 3H(2)O, space group P2(1), with a = 9.747(3) Angstrom, b = 21.002(8) Angstrom, c = 15.885(6) Angstrom, beta = 102.22(3)degrees, Z = 2, R = 13.6% for 2535 data observed >3 sigma(F). The observation of a helical conformation at Dbg suggests that the higher homologs in the alpha, alpha-dialkylated glycine series also have a tendency to stabilize peptide helices. (C) Munksgaard 1996.

Multiple conformational states in crystals and in solution in alpha gamma hybrid peptides. Fragility of the C-12 helix in short sequences

The conformational properties of foldamers generated from alpha gamma hybrid peptide sequences have been probed in the model sequence Boc-Aib-Gpn-Aib-Gpn-NHMe. The choice of alpha-aminoisobutyryl (Aib) and gabapentin (Gpn) residues greatly restricts sterically accessible coil formational space. This model sequence was anticipated to be a short segment of the alpha gamma C-12 helix, stabilized by three successive 4 -> 1 hydrogen bonds, corresponding to a backbone-expanded analogue of the alpha polypeptide 3(10)-helix. Unexpectedly, three distinct crystalline polymorphs were characterized in the solid state by X-ray diffraction. In one form, two successive C-12 hydrogen bonds were obtained at the N-terminus, while a novel C-17 hydrogen-bonded gamma alpha gamma turn was observed at the C-terminus. In the other two polymorphs, isolated C-9 and C-7 hydrogen-bonded turns were observed at Gpn (2) and Gpn (4). Isolated C-12 and C-9 turns were also crystallographically established in the peptides Boc-Aib-Gpn-Aib-OMe and Boc-Gpn-Aib-NHMe, respectively. Selective line broadening of NH resonances and the observation of medium range NH(i)<-> NH(i+2) NOEs established the presence of conformational heterogeneity for the tetrapeptide in CDCl3 solution. The NMR results are consistent with the limited population of the continuous C-12 helix conformation. Lengthening of the (alpha gamma)(n) sequences in the nonapeptides Boc-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Xxx (Xxx = Aib, Leu) resulted in the observation of all of the sequential NOEs characteristic of an alpha gamma C-12 helix. These results establish that conformational fragility is manifested in short hybrid alpha gamma sequences despite the choice of conformationally constrained residues, while stable helices are formed on chain extension.