Ensemble characterization of an intrinsically disordered FG-Nup peptide and its F>A mutant in DMSO-d6.

Intrinsically disordered proteins (IDP) lack a well-defined 3D-structure under physiological conditions, yet, the inherent disorder represented by an ensemble of conformation plays a critical role in many cellular and regulatory processes. Nucleoporins, or Nups, are the proteins found in the nuclear pore complex (NPC). The central pore of the NPC is occupied by Nups, which have phenylalanine-glycine domain repeats and are intrinsically disordered, and therefore are termed FG-Nups. These FG-domain repeats exhibit differing cohesiveness character and differ from least (FG) to most (GLFG) cohesive. The designed FG-Nup is a 25 AA model peptide containing a noncohesive FG-motif flanked by two cohesive GLFG-motifs (WT peptide). Complete NMR-based ensemble characterization of this peptide along with a control peptide with an F>A substitution (MU peptide) are discussed. Ensemble characterization of the NMR-determined models suggests that both the peptides do not have consistent secondary structures and continue to be disordered. Nonetheless, the role of cohesive elements mediated by the GLFG motifs is evident in the WT ensemble of structures that are more compact than the MU peptide. The approach presented here allows an alternate way to investigate the specific roles of distinct amino acid motifs that translate into the long-range organization of the ensemble of structures and in general on the nature of IDPs.

Structural analysis of the AICD central 12 residue peptide stretch and its interactions with metals and polyphenols, as a potential drug target for AD.

Structural analysis of the central 12 residue stretch of Amyloid precursor protein Intracellular Domain (AICD16-27: T-S-I-H-H-G-V-V-E-V-D-A) was carried out by NMR and homology modeling. Further, metal and polyphenol interactions were also carried out for these 12 residues stretch, as it contains two critical Histidine residues, which were observed to be perturbed via NMR. A full length 57 residues AICD model was generated via computational methods, to ascertain its overall conformation, as the entire structure was unavailable. An overlay of this AICD entire model with the full length Aß-42 structure matched well, implying similar properties. Docking studies with metals and polyphenols indicated involvement of the key Histidine residues highlighting their roles towards neurodegeneration and AD pathophysiology.Communicated by Ramaswamy H. Sarma.

Effect of ester chemical structure and peptide bond conformation in fragmentation pathways of differently metal cationized cyclodepsipeptides.

Fragmentation behavior of two classes of cyclodepsipeptides, isariins and isaridins, obtained from the fungus Isaria, was investigated in the presence of different metal ions using multistage tandem mass spectrometry (MS(n)) with collision induced dissociation (CID) and validated by NMR spectroscopy. During MS(n) process, both protonated and metal-cationized isariins generated product ions belonging to the identical 'b-ion' series, exhibiting initial backbone cleavage explicitly at the ß-ester bond. Fragmentation behavior for the protonated and metal-cationized acyclic methyl ester derivative of isariins was very similar. On the contrary, isaridins during fragmentation produced ions belonging to the 'b' or/and the 'y' ion series depending on the nature of interacting metal ions, due to initial backbone cleavages at the α-ester linkage or/and at a specific amide linkage. Interestingly, independent of the nature of the interacting metal ions, the product ions formed from the acyclic methyl ester derivative of isaridins belonged only to the 'y-type'. Complementary NMR data showed that, while all metal ions were located around the ß-ester group of isariins, the metal ion interacting sites varied across the backbone for isaridins. Combined MS and NMR data suggest that the different behavior in sequence specific charge-driven fragmentation of isariins and isaridins is predetermined because of the constituent ß-hydroxy acid residue in isariins and the cis peptide bond in isaridins.

Ring expansion of oxyglycals. Synthesis and conformational analysis of septanoside-containing trisaccharides.

Oxyglycals, derived from lactose and maltose, were expanded to trisaccharides through a ring expansion method. Trisaccharides with 6-7-5 and 6-7-6 ring sizes were prepared through the ring expansion method, with high diastereoselectivities, in each step of their synthesis. The NOE and ROESY NMR spectroscopies were used to assess the dipolar couplings within the trisaccharide. A computational study was undertaken, from which low energy conformations, as well as, dihedral angles that define the glycosidic linkages were identified.

Ni nanoparticle-confined covalent organic polymer directed diaryl-selenides synthesis.

The present work describes the preparation of a new covalent organic polymer (COP) and its application as a hetero support for diaryl selenides synthesis. A nitrogen rich COP (CGP) has been synthesized via SNAr reaction of cyanuric chloride with guanidinium hydrochloride. The successful confinement of COP with Ni nanoparticles through post-synthetic transformations (Ni@CGP) provides excellent catalytic activity for the transformation of aryl halides into diaryl selenides using elemental selenium powder. The synthetic transformations are well confirmed using various modern analytical and spectroscopic techniques which reveal high chemical and thermal durability. The N-rich framework of CGP fortifies the confinement of Ni NPs. Ni@CGP provides an efficient approach for diaryl selenides synthesis using a very cheap selenating reagent under water benign solvent conditions (DMSO : H2O) at room temperature with high reusability. Significantly, our work not only contributes the opportunity for developing economical and effective non-noble metal decorated COPs as heterogeneous catalysts, but also delivers an efficient approach to produce industrially important C-Se coupling products.

Solid-state NMR at natural isotopic abundance for the determination of conformational polymorphism - the case of designed ß-turn peptides containing di-prolines.

The proton double quantum-carbon single quantum correlation experiment has been applied to designed peptides in the solid state in natural isotopic abundance. Analogous to nOe studies in solution, through-space double-quantum connectivities have been exploited to obtain the cis-trans conformational polymorphism of diproline residues occurring at ß-turns in the peptides.

NMR structural analysis of a peptide mimic of the bridging sheet of HIV-1 gp120 in methanol and water.

gp120 is a subunit of the Env (viral envelope protein) of HIV-1. The protein consists of inner and outer domains linked by a bridging sheet. Several gp120 residues that bind the neutralizing antibody 17b as well as the cellular co-receptor CCR5 (CC chemokine receptor 5), are located in the bridging sheet. Peptides that mimic the 17b-binding regions of gp120 would be useful potential immunogens for the generation of neutralizing antibodies against HIV-1. Towards this end, a 26-residue, four-stranded beta-sheet peptide was designed on the basis of the structure of the bridging sheet, and its structure was characterized in methanol by NMR. In methanol, amide and alpha-proton resonances were well resolved and dispersed. A number of interstrand NOEs (nuclear Overhauser effects) were observed, providing good evidence for multiple turn beta-hairpin structure. NOEs also provided good evidence for all Xxx-D-Pro bonds in the trans configuration and all three turns formed by a two residue D-Pro-Gly segment to be of type II' turn. The structure conforms well to the designed four-stranded beta-sheet structure. Approx. 20% of the peptide was estimated to adopt a folded conformation in water, as evidenced by CD spectroscopy. This was consistent with smaller, but still significant, downfield shifts of C(alpha)H protons relative to random-coil values. A second peptide was designed with two disulphide bonds to further constrain the peptide backbone. While structured in methanol, this peptide, like the previous one, also exhibits only partial structure formation in water, as evidenced by CD spectroscopy.

A 1H-NMR study of bilirubin IX alpha solubilization by cholate micelles: application of nuclear Overhauser effects.

The solubilization of bilirubin IX alpha in aqueous solution by sodium cholate micelles has been examined by 270 MHz 1H-NMR spectroscopy. Incorporation of bilirubin into the micelles is accompanied by specific shifts of bilirubin vinyl and bridgehead protons and the C18 and C19 methyl groups of the steroid. The observed chemical shifts show a monotonic concentration dependence suggesting that changes in aggregation size are continuous. Nuclear Overhauser effects (NOE) have been shown to be a useful probe or micellization. A 4:1 cholate/bilirubin mixture has been investigated by difference NOE spectroscopy. The observation of intermolecular nuclear Overhauser effects between peripheral protons of bilirubin and cholate are diagnostic of spatially proximate groups. Inter-cholate nuclear Overhauser effects increase in magnitude upon bilirubin incorporation suggesting closer packing of steroid molecules on solubilization of the pigment. Intramolecular nuclear Overhauser effects observed for solubilized bilirubin are consistent with a compact intramolecularly hydrogen-bonded conformation resembling that determined for bilirubin in the solid state.

Chemistry in confined spaces: high-energy conformer of a piperidine derivative is favored within a water-soluble capsuleplex.

Propyloxy-substituted piperidine in solution adopts a conformation in which its alkoxy group is equatorially positioned. Surprisingly, two conformers of it that do not interconvert in the NMR time scale at room temperature have been found within an octa-acid capsule. The serendipitous finding of the axial conformer of propyloxy-substituted piperidine within a supramolecular capsule highlights the value of confined spaces in physical organic chemistry.

Natural abundant solid state NMR studies in designed tripeptides for differentiation of multiple conformers.

Solid state NMR (SSNMR) experiments on heteronuclei in natural abundance are described for three synthetically designed tripeptides Piv-(L)Pro-(L)Pro-(L)Phe-OMe (1), Piv-(D)Pro-(L)Pro-(L)Phe-OMe (2), and Piv-(D)Pro-(L)Pro-(L)Phe-NHMe (3). These peptides exist in different conformation as shown by solution state NMR and single crystal X-ray analysis (Chatterjee et al., Chem Eur J 2008, 14, 6192). In this study, SSNMR has been used to probe the conformations of these peptides in their powder form. The (13)C spectrum of peptide (1) showed doubling of resonances corresponding to cis/cis form, unlike in solution where the similar doubling is attributed to cis/trans form. This has been confirmed by the chemical shift differences of C(beta) and C(gamma) carbon of Proline in peptide (1) both in solution and SSNMR. Peptide (2) and (3) provided single set of resonances which represented all trans form across the di-Proline segment. The results are in agreement with the X-ray analysis. Solid state (15)N resonances, especially from Proline residues provided additional information, which is normally not observable in solution state NMR. (1)H chemical shifts are also obtained from a two-dimensional heteronuclear correlation experiment between (1)H--(13)C. The results confirm the utility of NMR as a useful tool for identifying different conformers in peptides in the solid state.

Tyrosine-heme ligation in heme-peptide complex: design based on conserved motif of catalase.

On the basis of evolutionary conservation of sequence in catalases, we have designed a heme-binding peptide (Ac-RLKSYTDTQISR12-(GGGG)-CRIVHC22-NH2) for the 'redox activity modulation' of heme. Heme-binding studies showed a blue-shifted Soret (369 nm) in the presence of TFE and a red-shifted Soret (418 nm) in the absence of TFE. These blue- and red-shifted Sorets suggest ligation through tyrosinate and histidine, respectively. This is the first designed peptide ligating to heme through tyrosine. NMR studies have confirmed that tyrosine ligation to heme in this heme-peptide complex occurs only in the presence of TFE. We suggest that TFE induces helicity in the peptide and brings the arginine and tyrosine in proximity, resulting in ionization of the phenolic side chain of tyrosine. In the absence of TFE, the unstructured peptide lacks the intra-molecular Arg(+)Tyr(-) ion pair, allowing heme binding to histidine. This peptide has significant peroxidase activity though it does not have catalase activity.

De novo design of DeltaF -containing heme-binding peptides.

The structural characterization of de novo designed metalloproteins together with determination of chemical reactivity can provide a detailed understanding of the relationship between protein structure and functional properties. Toward this goal, using the basic scaffold of 1pbz (Rosenblatt et al. (2003) Proc Natl Acad Sci U S A;100:13140) we have designed cyclic DeltaF-containing heme-binding peptides. The alpha- and beta-bands in UV-Vis spectroscopy are indicative of bis-His-ligated heme complex. Most of our DeltaF-containing peptides have more affinity to cobalt(III)Coproporphyrinate-I than heme because cobalt(III)Coproporphyrinate-I contains two additional propionate groups which can have salt bridge interactions with the lysine residues in the peptide. Helicity induction in peptide by DeltaF and aromatic interaction of DeltaF with heme have increased the heme affinity of CP-6-12pbz (cyclic peptide with substitutions of Ala at positions 6 and 12 by DeltaF; 905/mm) compared with 1pbz (279/mm). The nuclear magnetic resonance spectra are indicative of overall helical structure for CP-6-12pbz and CP-6-12pbz in complex with cobalt (III)Coproporphyrinate-I. The descending order of heme affinity in peptides (CP-6-12pbz > CP-12pbz > CP-5-12pbz) indicates that DeltaF at i + 3 or i - 3 from the central H9 favors heme binding but disrupts the same when placed at i - 4.

Identification and characterization of a library of microheterogeneous cyclohexadepsipeptides from the fungus Isaria.

Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have been identified and characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry (LC-ESIMS/MS). The isariins possess a beta-hydroxy acid residue and five alpha-amino acids, while isaridins contain a beta-amino acid, an alpha-hydroxy acid, and four alpha-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H]+ ions reveal clear diagnostic fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo, Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a hybrid alphabeta C11 turn, formed by the beta-hydroxy acid and glycine residues and a D Leu-L Ala type II' beta-turn. Additionally, the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.

Tryptophan-containing peptide helices: interactions involving the indole side chain.

Two designed peptide sequences containing Trp residues at positions i and i + 5 (Boc-Leu-Trp-Val-Ala-Aib-Leu-Trp-Val-OMe, 1) as well as i and i + 6 (Boc-Leu-Trp-Val-Aib-Ala-Aib-Leu-Trp-Val-OMe, 2) containing one and two centrally positioned Aib residues, respectively, for helix nucleation, have been shown to form stable helices in chloroform solutions. Structures derived from nuclear magnetic resonance (NMR) data reveal six and seven intramolecularly hydrogen-bonded NH groups in peptides 1 and 2, respectively. The helical conformation of octapeptide 1 has also been established in the solid state by X-ray diffraction. The crystal structure reveals an interesting packing motif in which helical columns are stabilized by side chain-backbone hydrogen bonding involving the indole Nepsilon1H of Trp(2) as donor, and an acceptor C=O group from Leu(6) of a neighboring molecule. Helical columns also associate laterally, and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules. The edge-to-face aromatic interactions between the indoles suggest a potential C-H...pi interaction involving the Czeta3H of Trp(2). Concentration dependence of NMR chemical shifts provides evidence for peptide association in solution involving the Trp(2) Nepsilon1H protons, presumably in a manner similar to that observed in the crystal.

Peptide hairpins with strand segments containing alpha- and beta-amino acid residues: cross-strand aromatic interactions of facing Phe residues.

The incporation of beta-amino acid residues into the strand segments of designed beta-hairpin leads to the formation of polar sheets, since in the case of beta-peptide strands, all adjacent carbonyl groups point in one direction and the amide groups orient in the opposite direction. The conformational analysis of two designed peptide hairpins composed of alpha/beta-hybrid segments are described: Boc-Leu-betaPhe-Val-(D)-Pro-Gly-Leu-betaPhe-Val-OMe (1) and Boc-betaLeu-Phe-betaVal-D-Pro-Gly-betaLeu-Phe-betaVal-OMe (2). A 500-MHz 1H-NMR (nuclear magnetic resonance) analysis in methanol supports a significant population of hairpin conformations in both peptides. Diagnostic nuclear Overhauser effects (NOEs) are observed in both cases. X-ray diffraction studies on single crystals of peptide 1 reveal a beta-hairpin conformation in both the molecules, which constitute the crystallographic asymmetric unit. Three cross-strand hydrogen bonds and a nucleating type II' beta-turn at the D-Pro-Gly segment are observed in the two independent molecules. In peptide 1, the betaPhe residues at positions 2 and 7 occur at the nonhydrogen-bonding position, with the benzyl side chains pointing on opposite faces of the beta-sheet. The observed aromatic centroid-to-centroid distances are 8.92 A (molecule A) and 8.94 A (molecule B). In peptide 2, the aromatic rings must occupy facing positions in antiparallel strands, in the NMR-derived structure. Peptide 1 yields a normal "hairpin-like" CD spectrum in methanol with a minimum at 224 nm. The CD spectrum of peptide 2 reveals a negative band at 234 nm and a positive band at 221 nm, suggestive of an exciton split doublet. Modeling of the facing Phe side chains at the hydrogen-bonding position of a canonical beta-hairpin suggests that interring separation is approximately 4.78 A for the gauche+ gauche- (g+ g-) rotamer. A previously reported peptide beta-hairpin composed of only alpha-amino acids, Boc-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe also exhibited an anomalous far-UV (ultraviolet) CD (circular dichroism) spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe 7 (C. Zhao, P. L. Polavarapu, C. Das, and P. Balaram, Journal of the American Chemical Society, 2000, Vol 122, pp. 8228-8231).

Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies.

Two cyclic peptide disulfides (Sequence: see text). (X = L-Tyr or L-Phe) have been synthesized as models for the 14-membered redox-active disulfide loop of glutaredoxin. 1H NMR studies at 270 MHz in chloroform solutions establish a type I beta-turn conformation for the Pro-X segment in both peptides, stabilized by a 4----1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type II beta-turn structures with -Pro-Tyr(Phe)- as the corner residues. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-S- n-omega* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.

Simultaneous observation of positive and negative nuclear Overhauser effects in oligopeptides due to segmental motion.

Nuclear Overhauser effect (NOE) studies of the symmetrical cystine peptides (Formula: see text) (n = 1-3) in dimethylsulfoxide, have resulted in the simultaneous observation of both positive and negative NOEs. Positive NOEs are observed on the Trp C2H and C4H protons of the indole ring upon irradiation of Trp C alpha H and C beta H2 resonances in the peptides where n = 1 and 2. Negative NOEs are observed between backbone NH and C alpha H protons. The magnitudes of the observed NOEs are sensitive to changes in molecular size and solvent viscosity. The results demonstrate that NOEs may be a useful probe of sidechain segmental motion in oligopeptides.

A designed beta-hairpin peptide.

A synthetic octapeptide, Boc-Leu-Val-Val-D-Pro-Gly-Leu-Val-Val-OMe (1) has been designed as a model for a beta-hairpin conformation. Circular dichroism spectra in various organic solvents reveal a single negative band at 214-217 nm consistent with beta-sheet structures. NMR studies in CDCl3 and C6D6 establish the solvent shielded nature of the Leu(1), Val(3), Leu(6) and Val (8) NH groups. Nuclear Overhauser effects are observed between Val(7) C alpha H and Val(2) C alpha H protons providing strong support for a beta-hairpin conformation. Several important diagnostic interresidue NOEs establish a Type II' beta-turn conformation for the D-Pro-Gly segment and extended conformations for the amino and carboxyl terminal tripeptide arms. The high solubility of the beta-hairpin peptide in organic solvents holds promise for the development of models for three and four stranded beta-sheets.

Conformational analysis of cyclolinopeptide A, a cyclic nonapeptide: nuclear Overhauser effect and energy minimization studies.

The conformation of cyclolinopeptide A [cyclo(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val)], a naturally occurring cyclic nonapeptide has been investigated in dimethylsulfoxide solution by 270 MHz 1H-nmr. A complete assignment of all C alpha H and NH resonances has been accomplished using two-dimensional correlated spectroscopy and nuclear Overhauser effects (NOEs). Analysis of interresidue NOEs and JHNC alpha H values permit construction of a molecular model for the cyclic peptide backbone. The crude model derived from nmr has been used as a starting point for energy minimization, which yields a refined structure largely compatible with nmr observations. The major features of the conformation of cyclolinopeptide A are a Type VI beta-turn centered at Pro(1)-Pro(2), with a cis peptide bond between these residues and a gamma-turn (C7 structure) centered at Ile(6). Two intramolecular hydrogen bonds Val(9) CO--Phe(3)NH (4----1) and Leu(5) CO--Ile(7)NH (3----1) are observed in the low-energy conformation. The limited solvent accessibility observed for the Val(9) and Leu(5) NH groups in the nmr studies are rationalized in terms of steric shielding.

Synthetic protein design: construction of a four-stranded beta-sheet structure and evaluation of its integrity in methanol-water systems.

The characterization of a four-stranded beta-sheet structure in a designed 26-residue peptide Beta-4 is described. The sequence of Beta-4 (Arg-Gly-Thr-Ile-Lys-(D)pro-Gly-Ile-Thr-Phe-Ala-(D)Pro-Ala-Thr-Val-Leu-P he-Ala-Val-(D)Pro-Gly-Lys-Thr-Leu-Tyr-Arg) was chosen such that three strategically positioned (D)Pro-Xxx segments nucleate type II' beta-turns, which facilitate hairpin extension. A four-stranded beta-sheet structure is determined in methanol from 500 MHz 1H NMR data using a total of 100 observed NOEs, 11 dihedral restraints obtained from vicinal JCalphaH-NH values and 10 hydrogen bonding constraints obtained from H/D exchange data. The observed NOEs provide strong evidence for a stable four-stranded sheet and a nonpolar cluster involving Ile8, Phe10, Val15 and Phe17. Circular dichroism studies in water-methanol mixtures provide evidence for melting of the beta-sheet structure at high water concentrations. NMR analysis establishes that the four-stranded sheet in Beta-4 is appreciably populated in 50% (v/v) aqueous methanol. In water, the peptide structure is disorganized, although the three beta-turn nuclei appear to be maintained.