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The cRGD-conjugated Aib-containing amphipathic helical peptide, MAP(Aib) derivative (PI), has been reported to be a useful carrier for siRNA delivery into cells. We have conducted a series of structure-activity relationship studies of the influence of the balance between hydrophobicity and basicity on the amphipathicity of PI, and synthesized peptides having a larger number of Lys residues than PI. Increasing the number of basic residues in the amphipathic helix suppressed the ability to deliver siRNA into cells. It was concluded that the balance between hydrophobicity and basicity in the PI helix was important for siRNA delivery into cells. Furthermore, the siRNA delivering ability of PI was specific to cancer cells, such as A549, U-87 MG, and WiDr cells, and was low in normal cells, namely, NIH3T3 cells. Next, we examined the potential of PI as a carrier for the delivery of microRNA-133b (miR-133b), which is known to be an anti-oncomiR. PI enhanced the delivery of miR-133b into WiDr cells, which resulted in the suppression of endogenous protein expression.

Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solvent-exposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by ∼3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (α-helix to π-helix) in order to precisely preserve the wild-type position and conformation of all hydrophobic residues, including Leu61. In addition to demonstrating that the hydrophobic side-chain of Leu61 is critically important for the stability of villin headpiece, our results are consistent with the notion that the precise interactions present within the hydrophobic core, rather than the hydrogen bonds that define the secondary structure, specify a protein's fold.

Extensive studies on the structure of collagen have revealed that the hydroxylation of Pro residues in a variety of model peptides with the typical (X-Y-Gly)nrepeats (X and Y: Pro and its analogues) represents one of the major factors influencing the stability of triple helices. While(2S,4R)-hydroxyproline (Hyp) at the position Y stabilizes the triple helix, (2S,4S)-hydroxyproline (hyp) at the X-position destabilizes the helix as demonstrated that the triple helix of (hyp-Pro-Gly)15 is less stable than that of (Pro-Pro-Gly)15 and that a shorter peptide (hyp-Pro-Gly)10 does not form the helix. To clarify the role of the hydroxyl group of Pro residues to play in the stabilization mechanism of the collagen triple helix, we synthesized and crystallized a model peptide (Pro-Hyp-Gly)4-(hyp-Pro-Gly)2-(Pro-Hyp-Gly)4 and analyzed its structure by X-ray crystallography and CD spectroscopy. In the crystal, the main-chain of this peptide forms a typical collagen like triple helix. The majority of hyp residues take down pucker with exceptionally shallow angles probably to relieve steric hindrance, but the remainders protrude the hydroxyl group toward solvent with the less favorable up pucker to fit in a triple helix. There is no indication of the existence of an intra-molecular hydrogen bond between the hydroxyl moiety and the carbonyl oxygen of hyp supposed to destabilize the triple helix. We also compared the conformational energies of up and down packers of the pyrrolidine ring in Ac-hyp-NMe2 by quantum mechanical calculations. © © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 111–121, 2012.

The F helix region of sperm whale apomyoglobin (apoMb) is disordered, undergoing conformational fluctuations between a folded helical conformation and one or more locally unfolded states. In order to examine the effects of F helix stabilization on the folding pathway of apoMb, we have introduced mutations to augment intrinsic helical structure in the F helix of the kinetic folding intermediate and to increase its propensity to fold early in the pathway, using predictions based on plots of the average area buried upon folding (AABUF) derived from the primary sequence. Two mutant proteins were prepared, a double mutant P88K/S92K (F2) and a quadruple mutant, P88K/A90L/S92K/A94L (F4). Whereas the AABUF for F2 predicts that the F helix will not fold early in the pathway, the F helix in F4 shows a significantly increased AABUF and is therefore predicted to fold early. Protection of amide protons by formation of hydrogen-bonded helical structure during the early folding events has been analyzed by pH-pulse labeling. Consistent with the AABUF prediction, many of the F helix residues for F4 are significantly protected in the kinetic intermediate but are not protected in the F2 mutant. F4 folds via a kinetically trapped burst phase intermediate that contains stabilized secondary structure in the A, B, F, G, and H helix regions. Rapid folding of the F helix stabilizes the central core of the misfolded intermediate and inhibits translocation of the H helix back to its native position, thereby decreasing the overall folding rate.

Apomyoglobin folds by a sequential mechanism in which the A, G, and H helix regions undergo rapid collapse to form a compact intermediate onto which the central portion of the B helix subsequently docks. To investigate the factors that frustrate folding, we have made mutations in the N-terminus of the B helix to stabilize helical structure (in the mutant G23A/G25A) and to promote native-like hydrophobic packing interactions with helix G (in the mutant H24L/H119F). The kinetic and equilibrium intermediates of G23A/G25A and H24L/H119F were studied by hydrogen exchange pulse labeling and interrupted hydrogen/deuterium exchange combined with NMR. For both mutants, stabilization of helical structure in the N-terminal region of the B-helix is confirmed by increased exchange protection in the equilibrium molten globule states near pH 4. Increased protection is also observed in the G-H turn region in the G23A/G25A mutant, suggesting that stabilization of the B-helix facilitates native-like interactions with the C-terminal region of helix G. These interactions are further enhanced in H24L/H119F. The kinetic burst phase intermediates of both mutants show increased protection, relative to wild type protein, of amides in the N-terminus of the B helix and in part of the E helix. Stabilization of the E helix in the intermediate is attributed to direct interactions between E helix residues and the newly stabilized N-terminus of helix B. Stabilization of native packing between the B and G helices in H24L/H119F also favors formation of native-like interactions in the GH turn and between the G and H helices in the ensemble of burst phase intermediates. We conclude that instability at the N-terminus of the B helix of apomyoglobin contributes to the energetic frustration of folding by preventing docking and stabilization of the E helix.

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Oc atom of Ser and Thr and the backbone carbonyl oxygen at position i 3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the b2-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding.

We have carried out a systematic analysis in order to evaluate whether Intra-Chain Disulfide Bridged Peptides (ICDBPs) observed in proteins of known three-dimensional structure adopt structurally similar conformations as they may correspond to structural/functional motifs. 406 representative ICDBPs comprising between 3 to 17 amino acid residues could be classified according to peptide sequence length and main-chain secondary structure conformation into 146 classes. ICDBPs comprising 6 amino acid residues are maximally represented in the Protein Data Bank. They also represent the maximum number of main-chain secondary structure conformational classes. Individual ICDBPs in each class represent different protein superfamilies and correspond to different amino acid sequences. We identified 145 ICDBP pairs that had not less-than 0.5 A root mean square deviation value corresponding to their equivalent peptide backbone atoms. We believe these ICDBPs represent structural motifs and possible candidates in order to further explore their structure/function role in the corresponding proteins. The common conformational classes observed for ICDBPs defined according to the main-chain secondary structure conformations; H (helix), B (residue in a isolated beta bridge), C (coil), E (extended beta strand), G (3(10) helix), I (pi helix), S (bend), T (hydrogen-bonded turn) were; "CHHH", "CTTC", "CSSS" and "CSSC" (for ICDBP length 4), "CSSCC" (length 5), "EETTEE", "CCSSCC", "CCSSSC" (length 6), "EETTTEE" (length 7), "EETTTTEE" (length 8), "EEEETTEEEE" (length 10), "EEEETTTEEEE" (length 11) and "EEEETTTTEEEE" (length 12).

The reversible manipulation of the helix screw sense in surface-grafted poly(beta-phenethyl-L-aspartate) (PPELA) films by means of external stimuli was investigated. Ringopening polymerization of beta-phenethyl-L-aspartate N-carboxyanhydride initiated from primary amino-functionalized silicon and quartz substrates results in surface-grafted PPELA films in which the end-grafted polypeptide chains have a right-handed alpha-helical conformation. Upon annealing of the film at 150 degrees C for 30 min, a helix screw sense inversion takes place and the grafted chains adopt C a left-handed T-helical conformation. In the solid state, this left-handed T-helical form is completely stable and cannot be changed by reheating and/orcooling. Upon immersion of the annealed grafted film in chloroform or other helicogenic solvents, the grafted polypeptide chains completely revert to their original right-handed alpha-helical form. Successive annealing and solvent treatment steps show that this helix sense inversion cycle can be repeated many times.

Calmodulin (CaM) binding to the AB module is crucial for multiple mechanisms governing the function of Kv7.2 (also known as KCNQ2) K(+) channel subunits, which mediate one of the main components of the non-inactivating K(+) M-current, a key controller of neuronal excitability. Structural analysis indicates that the CaM N-lobe engages with helix B, whereas the C-lobe anchors to the IQ site within helix A. Here, we report the identification of a new site between helices A and B that assists in CaM binding whose sequence is reminiscent of the TW helix within the CaM C-lobe anchoring site of SK2 K(+) channels (also known as KCNN2). Mutations that disrupt CaM binding within the TW site, helix B or helix A yield functional channels, whereas no function is observed when the TW site and helix A, or the TW site and helix B are mutated simultaneously. Our data indicate that the TW site is dispensable for function, contributes to the stabilization of the CaM-Kv7.2 complex and becomes essential when docking to either helix A or when helix B is perturbed.

Elongation factor Tu (EF-Tu) promotes binding of aminoacyl-tRNA to the A site of the ribosome. Here, we report the effects of mutations in helix D of EF-Tu and in the C-terminal domain of L7/12 on the kinetics of A-site binding. Reaction rates were measured by stopped-flow and quench-flow techniques. The rates of A-site binding were decreased by mutations at positions 144, 145, 148, and 152 in helix D of EF-Tu as well as at positions 65, 66, 69, 70, 73, and 84 in helices 4 and 5 of L7/12. The effect was due primarily to the lower association rate constant of ternary complex binding to the ribosome. These results suggest that helix D of EF-Tu is involved in an initial transient contact with helices 4 and 5 of L7/12 that promotes ternary complex binding to the ribosome. By analogy to the interaction of helix D of EF-Tu with the N-terminal domain of EF-Ts, the contact area is likely to consist of a hydrophobic patch flanked by two salt-bridges.

Secondary-structure elements (SSEs) play an important role in the folding of proteins. Identification of SSEs in proteins is a common problem in structural biology. A new method,ASSP(Assignment ofSecondaryStructure inProteins), using only the path traversed by the Cαatoms has been developed. The algorithm is based on the premise that the protein structure can be divided into continuous or uniform stretches, which can be defined in terms of helical parameters, and depending on their values the stretches can be classified into different SSEs, namely α-helices, 310-helices, π-helices, extended β-strands and polyproline II (PPII) and other left-handed helices. The methodology was validated using an unbiased clustering of these parameters for a protein data set consisting of 1008 protein chains, which suggested that there are seven well defined clusters associated with different SSEs. Apart from α-helices and extended β-strands, 310-helices and π-helices were also found to occur in substantial numbers.ASSPwas able to discriminate non-α-helical segments from flanking α-helices, which were often identified as part of α-helices by other algorithms.ASSPcan also lead to the identification of novel SSEs. It is believed thatASSPcould provide a better understanding of the finer nuances of protein secondary structure and could make an important contribution to the better understanding of comparatively less frequently occurring structural motifs. At the same time, it can contribute to the identification of novel SSEs. A standalone version of the program for the Linux as well as the Windows operating systems is freely downloadable and a web-server version is also available at http://nucleix.mbu.iisc.ernet.in/assp/index.php.

The structure of a single alanine-based ACE-AEAAAKEAAAKA-NH2 peptide in explicit aqueous solutions with mixed inorganic salts (NaCl and KCl) is investigated by using molecular simulations. The concentration of Na${}^{+}$, ${c}_{{\mathrm{Na}}^{+}}$, varies from 0.0$M$ to 1.0$M$, whereas the concentration of K${}^{+}$ is $1\ensuremath{-}{c}_{{\mathrm{Na}}^{+}}$. The simulated peptide is very sensitive to the change of concentration ratio between Na${}^{+}$ and K${}^{+}$. When the concentration ratio between Na${}^{+}$ and K${}^{+}$ is changed from 0.5/0.5, the structure of the peptide becomes loose or disordered. This specific phenomenon is confirmed via checking the changes of helix parameters and mapping the free energy along different coordinates. The higher normalized probability of forming direct and indirect salt bridges between residues Glu${7}^{\ensuremath{-}}$ and Lys11${}^{+}$ and the smallest probability of forming ringlike structures should be responsible for the stabilized helix structure in the 0.5 Na${}^{+}$/0.5 K${}^{+}$ solution. Furthermore, a noticeable conformational transition from an extended helix to an $\ensuremath{\alpha}$ helix is found in the 0.5 Na${}^{+}$/0.5 K${}^{+}$ solution, where a local ion cloud shows that some Na${}^{+}$ ions in the inner shells are still directly binding with the peptide, while K${}^{+}$ in the outer shells are moving into the inner shells, keeping the peptide in the collapsed state.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.

International audience; The hypothesis of structural alteration in transmembrane helices for signal transduction process is viewed by molecular dynamics simulation techniques. For the c-erbB-2 transmembrane domain involved in oncogenicity, the occurrence of conformational changes has been previously described as transition from the alpha to pi helix. This dynamical feature is thoroughly analyzed for the wild phenotype and oncogenic sequences from a series of 18 simulations carried out on one nanosecond time scale. We show that these structural events do not depend upon the conditions of simulations like force field or starting helix coordinates. We demonstrate that the oncogenic mutations Val659 Glu, Gln and Asp do not prevent the transition. Furthermore, we show that beta branched residues, in conjunction with Gly residues in the c-erbB-2 sequence, act as destabilizers for the alpha helix structure, pi deformations are tightly related to other local structural motifs found in soluble and membrane proteins. These structural alterations are discussed in term of structure-activity relationships for the c-erbB-2 activating mechanism mediated by transmembrane domain dimerization.

Models for the 3(10)-helix/coil and pi-helix/coil equilibria have been derived. The theory is based on classifying residues into helical or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation. The models for alpha-helix formation and 3(10)-helix formation have also been combined to describe a three-state equilibrium in which alpha-helical, 3(10)-helical, and coil conformations are populated. The results are compared with the modified Lifson-Roig theory for the alpha-helix/coil equilibrium. The comparison accounts for the experimental observations that 3(10)-helices tend to be short and pi-helices are not favored for any length. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices.

The i + 5-->i hydrogen bonded turn conformation (pi-turn) with the fifth residue adopting alpha L conformation is frequently found at the C-terminus of helices in proteins and hence is speculated to be a "helix termination signal." An analysis of the occurrence of i + 5-->i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C-terminus), using coordinates of 228 protein crystal structures determined by X-ray crystallography to better than 2.5 A resolution is reported in this paper. Of 486 detected pi-turn conformations, 367 have the (i + 4)th residue in alpha L conformation, generally occurring at the C-terminus of alpha-helices, consistent with previous observations. However, a significant number (111) of pi-turn conformations occur with (i + 4)th residue in alpha R conformation also, generally occurring in alpha-helices as distortions either at the terminii or at the middle, a novel finding. These two sets of pi-turn conformations are referred to by the names pi alpha L and pi alpha R-turns, respectively, depending upon whether the (i + 4)th residue adopts alpha L or alpha R conformations. Four pi-turns, named pi alpha L'-turns, were noticed to be mirror images of pi alpha L-turns, and four more pi-turns, which have the (i + 4)th residue in beta conformation and denoted as pi beta-turns, occur as a part of hairpin bend connecting twisted beta-strands. Consecutive pi-turns occur, but only with pi alpha R-turns. The preference for amino acid residues is different in pi alpha L and pi alpha R-turns. However, both show a preference for Pro after the C-termini. Hydrophilic residues are preferred at positions i + 1, i + 2, and i + 3 of pi alpha L-turns, whereas positions i and i + 5 prefer hydrophobic residues. Residue i + 4 in pi alpha L-turns is mainly Gly and less often Asn. Although pi alpha R-turns generally occur as distortions in helices, their amino acid preference is different from that of helices. Poor helix formers, such as His, Tyr, and Asn, also were found to be preferred for pi alpha R-turns, whereas good helix former Ala is not preferred. pi-Turns in peptides provide a picture of the pi-turn at atomic resolution. Only nine peptide-based pi-turns are reported so far, and all of them belong to pi alpha L-turn type with an achiral residue in position i + 4. The results are of importance for structure prediction, modeling, and de novo design of proteins.

Molecular dynamics simulations of a 29-residue peptide including the transmembrane domain of the V659G mutant of the c-erbB2 protein demonstrate important dynamical behavior. Although the alpha helix is the structure commonly assumed for transmembrane hydrophobic segments, we found that hydrogen bond rearrangements can occur, giving rise to a structural deformation termed pi bulge stabilized by successive hydrogen bonds of pi helix type. A series of simulations enables us to give a detailed description, at the atomic level, of the alpha helix->pi bulge transition. The major consequence of this deformation covering one and a half turn of helix results in a noticeable shift around the helix axis of the C-Terminal residues relatively to those of the N-terminus. Such a deformation closely related to structural motifs described in the literature, induces a change in the distribution of the residues along the helix faces which could modulate the protein activity mediated by a dimerization process.

Helix N-capping motifs often form hydrogen bonds with terminal amide groups which otherwise would be free. Also, without an amide hydrogen, proline (trans) is over-represented at helix N-termini (N1 position) because this naturally removes the need to hydrogen bond one terminal amide. However, the preference of cisPro, vis-a-vis helix N-termini, is not known. We show that cisPro (αR or PPII ) often appears at the N-cap position (N0) of helices. The N-cap cisPro(αR ) is associated with a six-residue sequence motif - X(-2) -X(-1) -cisPro-X(1) -X(2) -X(3) - with preference for Glu/Gln at X(-1) , Phe/Tyr/Trp at X(1) and Ser/Thr at X(3) . The motif, formed by the fusion of a helix and a type VIa β-turn, contains a hydrogen bond between the side chain of X(-1) and the side chain/backbone of X(3) , a α-helical hydrogen bond between X(-2) and X(2) and stacking interaction between cisPro and an aromatic residue at X(1) . NMR experiments on peptides containing the motif and its variants showed that local interactions associated with the motif, as found in folded proteins, were not enough to significantly tilt the cis/trans equilibrium towards cisPro. This suggests that some other evolutionary pressure must select the cisPro motif (over transPro) at helix N-termini. Database analysis showed that >C = O of the pre-cisPro(αR ) residue at the helix N-cap, directed opposite to the N→C helical axis, participates in long-range interactions. We hypothesize that the cisPro(αR ) motif is preferred at helix N-termini because it allows the helix to participate in long-range interactions that may be structurally and functionally important.

The specific cleavage of a DNA triple helix by FeII.bleomycin (BLM) is demonstrated. Triplex-specific cleavage was observed on both strands of the 32-base pair (bp) duplex at the duplex-triplex junctions. Strand scission products and alkali labile lesions were both formed. The strongest BLM cleavage site was located at the 5'-duplex-triplex junction, which is also the preferred triplex binding site of intercalating agents [Collier, D. A., Mergny, J.-L., Thuong, N. T., & Helene, C. (1991) Nucleic Acids Res. 19, 4219-4224]. The preference of BLM for the 5'-junction does not appear to derive from selective intercalative binding at this site. This is supported by the observation that phleomycin, which contains a thiazolinylthiazole moiety rather than a planar bithiazole ring system, exhibited the same selectivity of triplex cleavage as BLM. Cleavage of the triple helix by FeII.BLM was unaffected by concentrations of Mg2+ up to 5 mM, suggesting possible therapeutic applications of this novel DNA target. Molecular-modeling calculations of the triplex region suggested that dramatic variations in minor groove width and depth occur at the duplex-triplex junctions, particularly at the 5'-junction. Moreover, the minor groove at these sites was calculated to be somewhat shallower and wider than the minor groove of B-DNA. These results suggest that the preference of BLM for the duplex-triplex junctions derives from selective recognition of minor groove shape at these sites and thus reflects conformation-selective, rather than sequence-selective, DNA recognition by FeII.BLM.

A new homopolynucleotide triple helix, (dT)n.2(dA)n, detected by circular dichroism mixing curves, is the product of an endothermic reaction of (dA)n.(dT)n with (dA)n at moderate temperatures and high salt concentrations ([NaCl] > or = 2.6 M): (dA)n.(dT)n + (dA)n (dT)n.2(dA)n. At higher temperatures (dT)n.2(DA)n forms from the (dA)n.(dT)n duplex alone: 3[(dA)n.(dT)n] (dT)n.2(dA)n + (dA)n.2(dT)n. Upon further heating, (dT)n.2(dA)n is converted to the triplex (dA)n.2(dT)n: 2[(dT)n.2(dA)n] (dA)n.2(dT)n + 3(dA)n. (dT)n.2(dA)n forms owing to a favorable entropy change; delta Hm is unfavorable, ranging from 1 to 2.5 kcal mol-1, depending upon [NaCl]. The formation reaction is associated with a negative change in heat capacity. (dT)n.2(dA)n is an extremely weak complex with a free energy of stabilization, delta Go < or = 100 cal mol-1. Tm values of ultraviolet, circular dichroism, and differential scanning calorimetry transition curves for the formation of (dT)n.2(dA)n decrease with increasing [NaCl], reflecting, in part, the net uptake of cations. The values of (dTm/d ln a +/- )(delta Hm/RTm2) can be accounted for in terms of the charge spacing and cylindrical dimensions of the polynucleotides. The ionic strength dependence of this quantity is consistent with interaction of anions with (dAn). High concentrations of the anions Cl-, Br-, and ClO4- decrease the stability of (dT)n.2(dA)n according to the lyotropic series. The highly polarizable anion, ClO4-, entirely prevents the formation of (dT)n.2(dA)n. Phase diagrams of the (dA)n,(dT)n system in solutions of NaCl, NaBr, and NaClO4 are presented. A bonding scheme for (dT)n.2(dA)n is proposed, and implications of this work for Py.Pu.Pu triple helices are discussed.

α-Helices are the most abundant secondary structural motif in proteins (Kendrew et al., 1958, Pauling, Corey, & Branson, 1951) and an ideal linear helix can be completely described by a single valu...

The structure of helices within proteins is often distorted from the ideal linear topology. Curvature of the helix axis can be measured by determining the radius of a circle fit to the axis. Described here is a method of defining a curved path that places backbone atoms (usually Cα) equidistantly from the path. The variance in the distance of backbone atoms from the helix axis is minimised to produce the parametric equations that describe the intersection of a sphere and a plane. The geometric properties of the helix (including helix radius, radius of curvature, and pitch) can be readily obtained from these equations. The approach is applicable to any form of helix, can use any atom in the peptide to determine the axis, can be applied to any polypeptide including mixed α/β peptides, and does not rely on a regular spacing of peptide monomers in the polypeptide chain.

A DNA molecule is not a simple featureless rod but it takes a characteristic conformation depending on its base sequence as well as on its molecular environment. Each feature is likely to be recognized by a specific protein. In principle, the DNA molecule exists as a double helical B form, which is a majour target of DNA-binding proteins. Even in the B form it can bend with or without proteins. In addition, various DNA structures including a single-standed stable mini-hairpin, double helical Z and A forms, a triple helix and a quadruple helix have been observed.

Originally published in: Protein Folding Handbook. Part I. Edited by Johannes Buchner and Thomas Kiefhaber. Copyright © 2005 Wiley-VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3-527-30784-2 The sections in this article are Introduction Structure of the α-Helix Capping Motifs Metal Binding The 310-Helix The π-Helix Design of Peptide Helices Host–Guest Studies Helix Lengths The Helix Dipole Acetylation and Amidation Side Chain Spacings Solubility Concentration Determination Design of Peptides to Measure Helix Parameters Helix Templates Design of 310-Helices Design of π-helices Helix Coil Theory Zimm-Bragg Model Lifson-Roig Model The Unfolded State and Polyproline II Helix Single Sequence Approximation N- and C-Caps Capping Boxes Side-chain Interactions N1, N2, and N3 Preferences Helix Dipole 310- and π-Helices AGADIR Lomize-Mosberg Model Extension of the Zimm-Bragg Model Availability of Helix/Coil Programs Forces Affecting α-helix Stability Helix Interior Caps Phosphorylation Noncovalent Side-chain Interactions Covalent Side-chain interactions Capping Motifs Ionic Strength Temperature Trifluoroethanol pKa Values Relevance to Proteins Acknowledgments Keywords: alpha helix; pi helix; polyproline helix; helix design; helix stability

The critical Val/Glu mutation in the membrane spanning domain of the rat Neu receptor confers the ability for ligand-independent signaling and leads to increased dimerization and transforming ability. There is evidence that the two transmembrane interacting helices play a role in receptor activation by imposing orientation constraints to the intracellular tyrosine kinase domains. By using MD simulations we have attempted to discriminate between correct and improper helix- helix packing by examining the structural and energetic properties of preformed left-handed and right-handed structures in a fully hydrated DMPC bilayer. The best energetic balance between the residues at the helix- helix interface and the residues exposed to the lipids is obtained for helices in symmetrical left-handed interactions packed together via Glu side chain/Ala backbone interheli- cal hydrogen bonds. Analyses demonstrate the importance of the ATVEG motif in helix- helix packing and point to additional contacting residues necessary for association. Our findings, all consistent with experimental data, suggest that a symmetrical left-handed structure of the helices could be the transmembrane domain configuration that promotes receptor activation and transfor- mation. The present study may provide further insight into signal transduction mechanisms of the ErbB/Neu receptors. © 2005 Wiley Periodicals, Inc. Biopolymers 77: 184 -197, 2005

The most abundant helix type in proteins is the alpha-helix, accounting for about 31% of amino acid secondary structure states, while the 3(10)-helix accounts for about 4%. The pi-helix appears to be extremely rare and is considered to be unstable. Existing secondary structure definition methods find very few within the Protein Data Bank. Using an improved pi-helix definition algorithm to search a non-redundant subset of high-resolution and well-refined protein structures, we found that almost every tenth protein contained a pi-helix. This enabled us to show for the first time that the pi-helix has structural parameters that are different from the hypothesized model values. It also has distinctive amino acid preferences and it is conserved within functionally related proteins. Features that may contribute to the stability of the pi-helical structure have also been identified. In addition to hydrogen bonds, several other factors contribute to the stability of pi-helices. The pi-helix may have some functional advantages over other helical structures. Thus, we describe cases where the side chains of functionally important residues at every fourth position within a pi-helix could be aligned and brought close together in a way that would not be allowed by any other helix type.

Molecular dynamics simulations were carried out for three 13-residue peptides of the form AcNH-A-A-E-X-A-E-A-H-A-A-E-K-A-CONH(2) with X = A, F, and W. All three peptides exhibited unexpected dynamical behavior, undergoing a transition from an alpha-helical to a pi-helical structure in the course of 5-ns trajectories in aqueous solution. Analysis of peptide length, accessible surface, interaction energies, hydrogen bonding, and dihedral angles was consistent with alpha --> pi transitions at 2800, 500, and 800 ps for X = A, F and W, respectively. The transitions occurred sequentially and cooperatively, propagating from the C- to the N-terminus for X = A and W and from the center toward both termini for X = F. The time scale of the overall transition ranged from 300 to 500 ps. For all three peptides the backbone structural transition was accompanied by a concerted rearrangement of the charged side chains, including a 3 A increase in the distance between carboxylate groups of Glu-3 and Glu-6. During the transition the peptide backbone hydrogen-bonding patterns were disrupted at the interface between the alpha-helical and nascent pi-helical regions, with peptide groups forming water-bridged hydrogen bonds. The peptide structures exhibited significant fluidity, with individual residues sampling alpha-, pi-, and 3(10)-helical conformations, as well as a "coil" state, without any intramolecular hydrogen bonds. The studied peptides have been designed to form alpha-helices when incorporated in novel hemoprotein model compounds, peptide-sandwiched mesohemes, which consist of two identical peptides covalently attached to an Fe(III) mesoporphyrin [Liu, D., Williamson, D. A., Kennedy, M. L., Williams, T. D., Morton, M. M., and Benson, D. R. (1999) J. Am. Chem. Soc. 121, 11798-11812]. The possibility of adopting pi-helical structures by the constituent peptides may influence the properties of the hemoprotein models.

A search for the occurrence of the rare pi-helix was performed with Iditis from the Oxford Molecular Group upon the Protein Data Bank. In 8 of the 10 confirmed crystal structures that harbor the pi-helix, its unique conformation has been linked directly to the formation or stabilization of a specific binding site within the protein. In the discussion to follow, the role for each of these eight pi-helices will be addressed in regard to protein function. It is clear upon closer examination that the conformation of the pi-helix has evolved to provide unique structural features within a variety of proteins.

The ribosomal protein L9 has an unusual structure comprising two compact globular domains connected by a 34 residue alpha-helix. The middle 17 residues of the helix are exposed to solvent while the first seven pack against and form part of the N-terminal domain, and the last ten form part of the C-terminal domain. Here we report results which show that a peptide corresponding to the central helix of L9 is monomeric in aqueous solution and85% helical at 1 degrees C and 68(+/-7)% helical at 25 degrees C. This is considerably more helical than any other protein fragment studied to date. Another peptide corresponding to the middle 17 residues of the helix is monomeric and is 41(+/-4)% helical at 1 degrees C. Because the central helix has high intrinsic stability the globular N and C-terminal domains will likely be stabilized by their interactions with the helix. Therefore, the stability of the two terminal domains should not be completely independent because both domains gain stability from a shared structural element, the central helix. Also, the ability of the central helix to form native-like structure in isolation highlights a potential role for the helix in the early stages of the folding process.

The two structures reported in the article by Chin et al. [(2013). Acta Cryst. D69, 352–366] have been further refined and corrected.

The basic helix-loop-helix (bHLH) proteins constitute a family of eukaryotic transcription factors that have the ability to dimerize through a coiled-coil region, characterized by a series of highly conserved hydrophobic residues that form a helix-loop-helix structure. In this issue of Molecular Cell, Firulli et al. demonstrate that dimerization of the bHLH proteins, HAND1 and HAND2, is regulated by phosphorylation of residues located within the HLH domain.

PKB/Akt reveals a major role for helix C in the regulation of activity in the first structure of an AGC family protein kinase in its low-activity form.