Pi-turns in proteins and peptides: Classification, conformation, occurrence, hydration and sequence.

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.     

Tryptophan rich peptides: influence of indole rings on backbone conformation

Synthetic peptides with defined secondary structure scaffolds, namely hairpins and helices, containing tryptophan residues, have been investigated in this study to probe the influence of a large number of aromatic amino acids on backbone conformations. Solution NMR investigations of Boc-W-L-W-(D)P-G-W-L-W-OMe (peptide 1), designed to form a well-folded hairpin, clearly indicates the influence of flanking aromatic residues at the (D)Pro-Gly region on both turn nucleation and strand propagation. Indole-pyrrolidine interactions in this peptide lead to the formation of the less-frequent type I' turn at the (D)Pro-Gly segment and frayed strand regions, with the strand residues adopting local helical conformations. An analog of peptide 1 with an Aib-Gly turn-nucleated hairpin (Boc-W-L-W-U-G-W-L-W-OMe (peptide 2)) shows a preference for helical structures in solution, in both chloroform and methanol. Peptides with either one (Boc-W-L-W-U-W-L-W-OMe (peptide 3)) or two (Boc-U-W-L-W-U-W-L-W-OMe (peptide 4)) helix-nucleating Aib residues give rise to the well-folded helical conformations in the chloroform solution. The results are indicative of a preference for helical folding in peptides containing a large number of Trp residues. Investigation of a tetrapeptide analog of peptide 2, Boc-W-U-G-W-OMe (peptide 5), in solution and in the crystal state (by X-ray diffraction), also indicates a preference for a helical fold. Additionally, peptide 5 is stabilized in crystals by both aromatic interactions and an array of weak interactions. Examination of Trp-rich sequences in protein structures, however, reveals no secondary structure preference, suggesting that other stabilizing interactions in a well-folded protein may offset the influence of indole rings on backbone conformations.     

Light-induced oxidation of the telomeric G4 DNA in complex with Zn(II) tetracarboxymethyl porphyrin

Structure-specific ligands are convenient tools for the recognition, targeting or probing of non-canonical DNA structures. Porphyrin derivatives exhibit a preference for interaction with G-quadruplex (G4) structures over canonical duplex DNA and are able to cause photoinducible damage to nucleic acids. Here, we show that Zn(II) 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (ZnP1) interacts with different conformations of the telomeric sequence d(TAGGG(TTAGGG)₃) at submicromolar concentrations without any detectible disturbance of the particular fold. Among different folds, potassium (3+1) hybrid G4-structure. reveal the highest affinity to ZnP1. The pattern of guanine oxidation is specific for each telomeric DNA conformation and may serve as an additional tool for probing the G4 topology. The potassium (3+1) and parallel G4 conformations are more susceptible to light-induced oxidation than the sodium G4 conformation or double helix of the telomeric DNA. The major products of the guanine modifications are spiroiminodihydantoin (Sp) and 8-oxoguanine (8-oxoG). ZnP1-induced oxidation of guanines results in the structural rearrangement of parallel and (3+1) G4 conformations yielding an antiparallel-like G4 conformation. The mechanism of the observed light-induced conformational changes is discussed.     

Multiple conformations of NAD and NADH when bound to human cytosolic and mitochondrial aldehyde dehydrogenase

Crystallographic analysis revealed that the nicotinamide ring of NAD can bind with multiconformations to aldehyde dehydrogenase (ALDH) (Ni, L., Zhou, J., Hurley, T. D., and Weiner, H. (1999) Protein Sci. 8, 2784-2790). Electron densities can be defined for two conformations, neither of which appears to be compatible with the catalytic reaction. In one conformation, it would prevent glutamate 268 from functioning as a general base needed to activate the catalytic nucleophile, cysteine 302. In the other conformation, the nicotinamide is too far from the enzyme-substrate adduct for efficient hydride transfer. In this study, NMR and fluorescence spectroscopies were used to demonstrate that NAD and NADH bind to human liver cytosol and mitochondrial ALDH such that the nicotinamide samples a population of conformations while the adenosine region remains relatively immobile. Although the nicotinamide possesses extensive conformational heterogeneity, the catalyzed reaction leads to the stereospecific transfer of hydride to the coenzyme. Mobility allows the nicotinamide to move into position to be reduced by the enzyme-substrate adduct. Although the reduced nicotinamide ring retains mobility after NADH formation, the extent of the motion is less than that of NAD. It appears that after reduction the population of favored nicotinamide conformations shifts toward those that do not interfere with the ability of the enzyme to release the reaction product. In the case of the mitochondrial, but not the cytosolic, enzyme this change in conformational preference is promoted by the presence of Mg2+ ions. Coenzyme conformational mobility appears to be beneficial to catalysis by ALDH throughout the catalytic cycle.     

A model for the phosphorylation of the Ca2+ + Mg2+-activated ATPase by phosphate.

We have shown that changes in fluorescence intensity for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate following the addition of Ca2+ can give the ratio of the two conformations (E1 and E2) of the ATPase. We show that the fluorescence response to Ca2+ is unaffected by Mg2+, phosphate or K+, implying that these ions bind equally well to the E1 and E2 conformations. A model is presented for phosphorylation of the ATPase by phosphate as a function of pH, Mg2+, K+ and Ca2+.     

Fast and accurate computation of the 13C chemical shifts for an alanine-rich peptide

The purpose of this work is, first, to present a fast and accurate technique to compute Boltzmann-averaged values of the quantum-chemical 13C chemical shifts for each amino acid in oligopeptides, demonstrated here by an application to the peptide Ac-XXAAAAAAAOO-NH2 (where X denotes diaminobutyric acid, A is alanine, and O is ornithine) [XAO] and, second, to discuss the capability of the 13Calpha and 13Cbeta chemical shifts to distinguish the PP(II) conformation from the alpha-helix and statistical-coil conformations. Use is made of a combination of approaches, summarized as follows: (1) derivation of an ensemble of conformations by using a molecular mechanics technique; (2) use of a clustering procedure to form families and build a reduced set of conformations consisting of the lowest-energy conformations of each family, and (3) computation of the 13C chemical shifts for the lowest-energy conformations of each family, using a quantum-chemical approach that treats a selected residue, or group of residues, with a 6-311+G(2d,p) locally-dense basis set, while the remaining residues in the sequence are treated with a 3-21G basis set. The whole procedure is quite accurate and speeds up the computation of the Boltzmann-averaged values of the 13C-chemical shifts by several orders of magnitude. The present application sheds some light on the conformational preference for alanine and non-alanine residues to occupy the PP(II) helical region of the Ramachandran map.     

Spectroscopic studies of oligomers containing 2,5-trans furanoid sugar amino acids

Sugar amino acids and their oligomers, known as carbopeptoids, are commonly studied as foldamers. However, study of their conformational preference is often challenging when the adopted conformations are extended and/or disordered. This study is the first to explore the disordered nature of such carbopeptoids by utilizing a family of 2,5-trans carbopeptoids. An array of spectroscopic techniques has been used to investigate the conformational preference of these carbopeptoids. However, using this data alone it has not been possible to assign conformational preference as an ordered extended conformation or as a disordered family of closely related conformations. Computational methods need to be employed to achieve reliable interpretation of the spectroscopic data.     

Positional preference of proline in alpha-helices.

Conformational free energy calculations have been carried out for proline-containing alanine-based pentadecapeptides with the sequence Ac-(Ala)n-Pro-(Ala)m-NHMe, where n + m = 14, to figure out the positional preference of proline in alpha-helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the alpha-helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N-terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N' (Ncap - 1), and C' (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X-ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing alpha-helical conformations, which, however, provides the favored hydration of the corresponding N-H and C=O groups. The highest preference of proline at the beginning of alpha-helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of alpha-helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of alpha-helix by proline. The average free energy change for the substitution of Ala by Pro in a alpha-helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of approximately 4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme.     

Molecular mechanisms of imidazole and benzene ring binding in proteins

Aromatic bonds of amino acid radicals play an important role in arrangement of protein primary structure. Previously, the existence of a number of preferable conformations of aromatic dimers was shown theoretically and experimentally, the best known of which are parallel-displaced and perpendicular T conformations. To reveal principles that define preference of various conformations for His-His and Phe-His dimers, non-empirical quantum-chemical calculations of diimidazole and benzene-imidazole were carried out. Calculations were performed using the 6-31G** basis with account for electronic correlations in frames of MP2 and MP4 methods of perturbation theory. Comparative analysis of energetic and geometric parameters of the systems points to the preference of stacking contact or classical hydrogen bond in diimidazole. On the contrary, T conformation is maximally advantageous for benzene-imidazole.     

An RNA aptamer that recognizes a specific conformation of the protein calsenilin

The generation of molecules that selectively recognize specific conformations of a protein is an important component of the elucidation protein function. We have used SELEX (Systematic Evolution of Ligands by EXponential enrichment) technology to produce aptamers that bind in a conformationally selective manner to calsenilin, which involved in Ca²⁺-mediated apoptotic signaling. Since the conformations of calsenilin are quite different in the presence and absence of Ca²⁺, aptamers were selected against the dimeric protein both under calcium-bound and calcium-free conditions. We have found that aptamer-12 selectively binds to the dimeric form of the protein in the presence of calcium ion, while the binding of aptamer-2 does not discriminate between the Ca²⁺ bound and unbound protein. Data obtained from biochemical and biophysical experiments suggest that a dominant conformation of calcium-bound calsenilin exists in one dominant conformation and that one aptamer can be generated to recognize this conformation. In addition, observation made in this effort that aptamers selected against the two different conformations of calsenilin have different characteristics suggest that aptamers can serve as a plausible tool for recognizing various conformations of proteins, even those caused by interactions with small molecules or ions such as Ca²⁺.     

Secondary structure inducing potential of beta-amino acids: torsion angle clustering facilitates comparison and analysis of the conformation during MD trajectories

While numerous examples of beta-peptides--exclusively composed of beta-amino acids--have been investigated during the past decade, there are only few reports on the conformational preference of a single beta-amino acid when incorporated into a cyclopeptide. The conformational bias of beta-amino acids on the secondary structure of cyclopeptides has been investigated by NMR spectroscopy in combination with distance geometry (DG) and molecular dynamics (MD) calculations using experimental constraints. The atomic coordinate RMSD criterion usually employed for clustering of conformations after DG and MD calculations does not necessarily group similar peptide conformations, as there is an insufficient correlation between atomic coordinates and torsion angles. To improve on this shortcoming and to eliminate any arbitrary decisions during this process, a torsion angle clustering procedure has been implemented. For the cyclic pentapeptides cyclo-(-Val-beta-Hala-Phe-Leu-Ile-) 1 and cyclo-(-Ser-Pro-Leu-beta-Hasn-Asp-) 3, the beta-amino acid is found in the central position of an extended gamma-turn (pseudo gamma-turn, Psigamma-turn), while the beta-Hpro residue in the cyclic hexapeptide cyclo-(-Ser-beta-Hpro-Leu-Asn-Ile-Asp-) 5 preferentially occupies position i+1 of a pseudo beta-turn (Psibeta-turn). These results further corroborate the hypothesis of beta-amino acids being reliable inducers of secondary structure in cyclic penta- and hexapeptides. They can be employed in the de novo design of biologically active cyclopeptides in pharmaceutical research, since the three-dimensional presentation of pharmacophoric groups in the side chains can be tailored by incorporation of beta-amino acids in strategic sequential positions.     

Role of azaamino acid residue in beta-turn formation and stability in designed peptide.

The structural perturbation induced by C(alpha)-->N(alpha) exchange in azaamino acid-containing peptides was predicted by ab initio calculation of the 6-31G* and 3-21G* levels. The global energy-minimum conformations for model compounds, For-azaXaa-NH2 (Xaa=Gly, Ala, Leu) appeared to be the beta-turn motif with a dihedral angle of phi= +/- 90 degrees, psi=0 degrees. This suggests that incorporation of the azaXaa residue into the i+2 position of designed peptides could stabilize the beta-turn structure. The model azaLeu-containing peptide, Boc-Phe-azaLeu-Ala-OMe, which is predicted to adopt a beta-turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using 1H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (phi i+1, psi i+1) (phi i+2, psi i+2)] of the Phe-azaLeu fragment in the model peptide, Boc-Phe-azaLeu-Ala-OMe, as [(-59 degrees, 127 degrees) (107 degrees, -4 degrees)]. This solution conformation supports a betaII-turn structural preference in azaLeu-containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i+2 position in synthetic peptides is expected to provide a stable beta-turn formation, and this could be utilized in the design of new peptidomimetics adopting a beta-turn scaffold.     

Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H fold

Three-way junctions (3H) are the simplest and most commonly occurring branched nucleic acids. They consist of three double helical arms (A to C), connected at the junction point, with or without a number of unpaired bases in one or more of the three different strands. Three-way junctions with two unpaired bases in one strand (3HS2) have a high tendency to adopt either of two alternative stacked conformations in which two of the three arms A, B and C are coaxially stacked, i.e. A/B-stacked or A/C-stacked. Empirical stacking rules, which successfully predict for DNA 3HS2 A/B-stacking preference from sequence, have been extended to A/C-stacked conformations. Three novel DNA 3HS2 sequences were designed to test the validity of these extended stacking rules and their conformational behavior was studied by solution NMR. All three show the predicted A/C-stacking preference even in the absence of multivalent cations. The stacking preference for both classes of DNA 3HS2 can thus be predicted from sequence. The high-resolution NMR solution structure for one of the stacked 3HS2 is also reported. It shows a well-defined local and global structure defined by an extensive set of classical NMR restraints and residual dipolar couplings. Analysis of its global conformation and that of other representatives of the 3H family, shows that the relative orientations of the stacked and non-stacked arms, are restricted to narrow regions of conformational space, which can be understood from geometric considerations. Together, these findings open up the possibility of full prediction of 3HS2 conformation (stacking and global fold) directly from sequence.     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ +K+)-dependent ATPase

Quercetin inhibited a dog kidney (Na+ + K+)-ATPase preparation without affecting Km for ATP or K0.5 for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the (Na+ + K+)-ATPase activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the Km for ATP and the K0.5 for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on Vmax, Km for substrate, and K0.5 for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations.     

Hydration of the left spiral of the poly-L-proline type. Study by the Monte Carlo method]

The paper exhibits results of hydration shell Monte Carlo calculations in poly-L-proline II and extended helix conformation and in alpha-helical and beta-structural conformations for comparison. It was found that left-handed helix of poly-L-proline II type as well as epsilon-helix are characterized by very favorable hydration. Therefore this conformation has preference as compared to other standard conformations of the main polypeptide chain. This determined inevitability of cold denaturation of protein.     

Fluorine-19 NMR studies of glucosyl fluoride transport in human erythrocytes.

Fluorine-19 magnetization transfer studies have been used to measure the transport rate of glucopyranosyl fluorides under equilibrium exchange conditions. Although rate constants and permeabilities could be determined for beta-D-glucopyranosyl fluoride, the exchange rate for alpha-D-glucopyranosyl fluoride was found to be too slow for determination using this method. The time-dependent decomposition of the beta-glucopyranosyl fluoride also limits the accuracy of the numerical results for this species; however, it is clear that the permeabilities of the alpha and beta forms differ significantly, i.e., P beta > P alpha. This observation is in contrast to recent observations for n-fluoro-n-deoxyglucose, for which P alpha > P beta for n = 2, 3, 4, or 6. The difference can be explained in terms of a simple alternating conformation model in which one of the conformations (with an external sugar-binding site) exhibits a preference for the beta form of the molecule, while the second conformation (with an internal sugar binding site) exhibits a preference for the alpha form. Fluorine/hydroxyl substitutions unmask these preferences by selectively reducing the binding to one of the conformations, depending on the specific site of fluorination.     

Conformational stability of a thrombin-binding peptide derived from the hirudin C-terminus.

The COOH-terminal region of hirudin represents an independent functional domain that binds to an anion-binding exosite of thrombin and inhibits the interaction of thrombin with fibrinogen and regulatory proteins in blood coagulation. The thrombin-bound structure of the peptide fragment, hirudin 55-65, has been determined by use of transferred NOE spectroscopy [Ni, F., Konishi, Y., & Scheraga, H. A. (1990) Biochemistry 29, 4479-4489]. The stability of the thrombin-bound conformation has been characterized further by a combined NMR and theoretical analysis of the conformational ensemble accessible by the hirudin peptide. Medium- and long-range NOE's were found for the free hirudin peptide in aqueous solution and in a mixture of dimethyl sulfoxide and water at both ambient (25 degrees C) and low (0 degrees C) temperatures, suggesting that ordered conformations are highly populated in solution. The global folding of these conformations is similar to that in the thrombin-bound state, as indicated by NOE's involving the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Residues Glu(61), Glu(62), Tyr(63), and Leu(64) all contain approximately 50% of helical conformations calculated from the ratio of the sequential dNN and d alpha N NOE's. Among the helical ensemble, active 3(10)-helical conformations were found by an analysis of the medium-range [(i,i+2) and (i,i+3)] NOE's involving the last six residues of the peptide. An analysis of the side-chain rotamers revealed that, upon binding to thrombin, there may be a rotation around the alpha CH-beta CH bond of Ile(59) such that Ile(59) adopts a gauche- (chi 1 = +60) conformation in contrast to the highly populated trans (chi 1 = -60) found for Ile(59) in the free peptide. However, the thrombin-bound conformation of the hirudin peptide is still an intrinsically stable conformer, and the preferred conformational ensemble of the peptide contains a large population of the active conformation. The apparent preference for a gauche- (chi 1 = +60) side-chain conformation of Ile(59) in the bound state may be explained by the existence of a positively charged arginine residue among the hydrophobic residues in the thrombin exosite.     

Torsion angle based design of peptidomimetics: a dipeptidic template adopting beta-I turn (Ac-Aib-AzGly--NH(2)).

We have attempted to design a model dipeptide (acetyl dipeptide amide, Ac-CA1-CA2--NH(2)) that can adopt specifically typical torsion angles of the beta-I turn (phi(i+1), psi(i+1), phi(i+2), psi(i+2)=-60 degrees, -30 degrees, -90 degrees, 0 degrees ). The key of the design is the combination of constrained amino acids that prefer to adopt the desired torsion angles. We chose Aib (aminoisobutyric acid) as the first residue of which phi and psi angles must be -60 degrees and -30 degrees, respectively. Then, we selected an azaamino acid as the second residue since previous studies have indicated that they prefer to adopt +/-90 degrees of phi angle and 0 degrees or 180 degrees of psi angle. The conformational preference of the resulting Ac-Aib-AzGly--NH(2) is investigated using ab initio methods. The conformations implying beta-I and beta-I' turns are energetically most favorable, as we expected. Thus, we synthesized the designed molecule on the solid phase considering the future generation of combinatorial libraries using an automatic peptide synthesizer. Then, NMR spectroscopy was carried out to confirm their conformational preference in solution was carried out. The results indicated that the Ac-Aib-AzGly--NH(2) adopt beta-I or beta-I' turns in solution forming an intramolecular hydrogen bonding between Ac--C(O) and terminal NH(2). We believe that such a small peptidomimetic template is highly useful for the design of drug candidates and molecular devices.     

Helix, sheet, and polyproline II frequencies and strong nearest neighbor effects in a restricted coil library

A central issue in protein folding is the degree to which each residue's backbone conformational preferences stabilize the native state. We have studied the conformational preferences of each amino acid when the amino acid is not constrained to be in a regular secondary structure. In this large but highly restricted coil library, the backbone preferentially adopts dihedral angles consistent with the polyproline II conformation rather than alpha or beta conformations. The preference for the polyproline II conformation is independent of the degree of solvation. In conjunction with a new masking procedure, the frequencies in our coil library accurately recapitulate both helix and sheet frequencies for the amino acids in structured regions, as well as polyproline II propensities. Therefore, structural propensities for alpha-helices and beta-sheets and for polyproline II conformations in unfolded peptides can be rationalized solely by local effects. In addition, these propensities are often strongly affected by both the chemical nature and the conformation of neighboring residues, contrary to the Flory isolated residue hypothesis.