Heterodetic bicyclic decapeptide cyclo (Glu1-Leu2-Pro3-Gly4-Ser5-Ile6-Pro7- Ala8) cyclo-(1 gamma-5 beta) Phe9-Gly10. Synthesis and 2-D NMR conformational analysis

Bicyclic peptides are useful model molecules that can mimic the constrained local folding of a great number of natural peptides and proteins, such as ionophoric peptides, enzyme active site, and ligand-receptor active site. The synthesis of the bicyclic title compound with the liquid phase method is described with experimental details. Of particular interest is the heterodetic closure of the second ring. The peptide showed a complexing activity with metal cations like Ba2+, Ca2+, and Mg2+. The free bicyclic peptide conformation in solution has been studied by means of NMR spectroscopy and a plausible structure model worked out with model building on NMR constraints is proposed.     

Solution structure of a D,L-alternating oligonorleucine as a model of double-stranded antiparallel beta-helix

Conformational characteristics of alternating D,L linear peptides are of particular interest because of their capacity to form transmembrane channels with different transport properties, as some natural antibiotics do. Single- and double-stranded beta-helical structures are common for alternating D,L peptides. The stability of the beta-helix depends on several structural factors, such as the backbone peptide length, type and position of side chains, and nature of terminal groups. The NMR and molecular dynamics solution conformation of a synthetic alternating D,L-oligopeptide with 15 norleucines (XVMe) has been used as a model to get insight in to the conformational features of double-stranded beta-helix structures. The NH chemical shift values (delta(NH)) and long-range nuclear Overhauser effects (NOE) cross peaks, in particular interstrand connectivities, clearly point to an antiparallel double-stranded beta-helix for the XVMe major conformation in solution. An extensive set of distances (from NOE cross peaks) and H-bonds (from delta(NH)) has been included in the molecular dynamics calculations. The experimental NMR data and theoretical calculations clearly indicate that the most probable conformation of XVMe in solution is a double-strand antiparallel beta(5.6) increasing decreasing-helix structure.     

A polynomial-time algorithm for de novo protein backbone structure determination from nuclear magnetic resonance data.

We describe an efficient algorithm for protein backbone structure determination from solution Nuclear Magnetic Resonance (NMR) data. A key feature of our algorithm is that it finds the conformation and orientation of secondary structure elements as well as the global fold in polynomial time. This is the first polynomial-time algorithm for de novo high-resolution biomacromolecular structure determination using experimentally recorded data from either NMR spectroscopy or X-ray crystallography. Previous algorithmic formulations of this problem focused on using local distance restraints from NMR (e.g., nuclear Overhauser effect [NOE] restraints) to determine protein structure. This approach has been shown to be NP-hard, essentially due to the local nature of the constraints. In practice, approaches such as molecular dynamics and simulated annealing, which lack both combinatorial precision and guarantees on running time and solution quality, are used routinely for structure determination. We show that residual dipolar coupling (RDC) data, which gives global restraints on the orientation of internuclear bond vectors, can be used in conjunction with very sparse NOE data to obtain a polynomial-time algorithm for structure determination. Furthermore, an implementation of our algorithm has been applied to six different real biological NMR data sets recorded for three proteins. Our algorithm is combinatorially precise, polynomialtime, and uses much less NMR data to produce results that are as good or better than previous approaches in terms of accuracy of the computed structure as well as running time.     

High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA

We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2′-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined.     

Building native protein conformation from NMR backbone chemical shifts using Monte Carlo fragment assembly

We have been analyzing the extent to which protein secondary structure determines protein tertiary structure in simple protein folds. An earlier paper demonstrated that three-dimensional structure can be obtained successfully using only highly approximate backbone torsion angles for every residue. Here, the initial information is further diluted by introducing a realistic degree of experimental uncertainty into this process. In particular, we tackle the practical problem of determining three-dimensional structure solely from backbone chemical shifts, which can be measured directly by NMR and are known to be correlated with a protein's backbone torsion angles. Extending our previous algorithm to incorporate these experimentally determined data, clusters of structures compatible with the experimentally determined chemical shifts were generated by fragment assembly Monte Carlo. The cluster that corresponds to the native conformation was then identified based on four energy terms: steric clash, solvent-squeezing, hydrogen-bonding, and hydrophobic contact. Currently, the method has been applied successfully to five small proteins with simple topology. Although still under development, this approach offers promise for high-throughput NMR structure determination.     

Review: model peptides and the physicochemical approach to beta-amyloids

beta-Amyloid peptides are the main protein components of neuritic plaques and may be important in the pathogenesis of Alzheimer's Disease. The determination of the structure of beta-amyloid fibrils poses a challenge because of the limited solubility of beta-amyloid peptides and the noncrystalline nature of fibrils formed from these peptides. In this paper, we describe several physicochemical approaches which have been used to examine fibrils and the fibrillogenesis of peptide models of beta-amyloid. Recent advances in solid state NMR, such as the DRAWS pulse sequence, have made this approach a particularly attractive one for peptides such as beta-amyloid, which are not yet amenable to high-resolution solution phase NMR and crystallography. The application of solid state NMR techniques has yielded information on a model peptide comprising residues 10-35 of human beta-amyloid and indicates that in fibrils, this peptide assumes a parallel beta-strand conformation, with all residues in exact register. In addition, we discuss the use of block copolymers of Abeta peptides and polyethylene glycol as probes for the pathways of fibrillogenesis. These methods can be combined with other new methods, such as high-resolution synchrotron X-ray diffraction and small angle neutron and X-ray scattering, to yield structural data of relevance not only to disease, but to the broader question of protein folding and self-assembly.     

Solid-state NMR studies of the structure and mechanisms of proteins

Magic-angle spinning solid-state NMR experiments are well suited to investigating the structures and mechanisms of important proteins that are inaccessible to X-ray crystallography and solution NMR spectroscopy, including membrane proteins and disease-related protein aggregates. Good progress has been made in the development of methods for the complete structure determination of small (<20 kDa) solid proteins using uniformly 13C, 15N-labeled samples. Studies of selectively labeled proteins focusing on labeled active sites have yielded insights into the mechanisms of enzymes and of membrane proteins involved in energy and signal transduction. Studies of selectively labeled synthetic peptides have yielded structural models for biomedically important systems, including amyloid fibrils and surface-associated peptides involved in biomineralization and cell adhesion. Novel NMR and biochemical methods are being developed to target solid-state NMR experiments within large proteins and whole cells. These approaches are being used to investigate mechanisms of transmembrane signaling by membrane receptors and to characterize binding interactions between antibiotics and bacterial cell walls. Thus, solid-state NMR is proving to be a valuable biophysical tool for probing structure and dynamics in a wide range of biomolecules.     

Local interactions drive the formation of nonnative structure in the denatured state of human alpha-lactalbumin: a high resolution structural characterization of a peptide model in aqueous solution.

There are a small number of peptides derived from proteins that have a propensity to adopt structure in aqueous solution which is similar to the structure they possess in the parent protein. There are far fewer examples of protein fragments which adopt stable nonnative structures in isolation. Understanding how nonnative interactions are involved in protein folding is crucial to our understanding of the topic. Here we show that a small, 11 amino acid peptide corresponding to residues 101-111 of the protein alpha-lactalbumin is remarkably structured in isolation in aqueous solution. The peptide has been characterized by 1H NMR, and 170 ROE-derived constraints were used to calculate a structure. The calculations yielded a single, high-resolution structure for residues 101-107 that is nonnative in both the backbone and side-chain conformations. In the pH 6.5 crystal structure, residues 101-105 are in an irregular turn-like conformation and residues 106-111 form an alpha-helix. In the pH 4.2 crystal structure, residues 101-105 form an alpha-helix, and residues 106-111 form a loopike structure. Both of these structures are significantly different from the conformation adopted by our peptide. The structure in the peptide model is primarily the result of local side-chain interactions that force the backbone to adopt a nonnative 310/turn-like structure in residues 103-106. The structure in aqueous solution was compared to the structure in 30% trifluoroethanol (TFE), and clear differences were observed. In particular, one of the side-chain interactions, a hydrophobic cluster involving residues 101-105, is different in the two solvents and residues 107-111 are considerably more ordered in 30% TFE. The implications of the nonnative structure for the folding of alpha-lactalbumin is discussed.     

Solution NMR studies of amphibian antimicrobial peptides: Linking structure to function?

The high-resolution three-dimensional structure of an antimicrobial peptide has implications for the mechanism of its antimicrobial activity, as the conformation of the peptide provides insights into the intermolecular interactions that govern the binding to its biological target. For many cationic antimicrobial peptides the negatively charged membranes surrounding the bacterial cell appear to be a main target. In contrast to what has been found for other classes of antimicrobial peptides, solution NMR studies have revealed that in spite of the wide diversity in the amino acid sequences of amphibian antimicrobial peptides (AAMPs), they all adopt amphipathic α-helical structures in the presence of membrane-mimetic micelles, bicelles or organic solvent mixtures. In some cases the amphipathic AAMP structures are directly membrane-perturbing (e.g. magainin, aurein and the rana-box peptides), in other instances the peptide spontaneously passes through the membrane and acts on intracellular targets (e.g. buforin). Armed with a high-resolution structure, it is possible to relate the peptide structure to other relevant biophysical and biological data to elucidate a mechanism of action. While many linear AAMPs have significant antimicrobial activity of their own, mixtures of peptides sometimes have vastly improved antibiotic effects. Thus, synergy among antimicrobial peptides is an avenue of research that has recently attracted considerable attention. While synergistic relationships between AAMPs are well described, it is becoming increasingly evident that analyzing the intermolecular interactions between these peptides will be essential for understanding the increased antimicrobial effect. NMR structure determination of hybrid peptides composed of known antimicrobial peptides can shed light on these intricate synergistic relationships. In this work, we present the first NMR solution structure of a hybrid peptide composed of magainin 2 and PGLa bound to SDS and DPC micelles. The hybrid peptide adopts a largely helical conformation and some information regarding the inter-helix organization of this molecule is reported. The solution structure of the micelle associated MG2-PGLa hybrid peptide highlights the importance of examining structural contributions to the synergistic relationships but it also demonstrates the limitations in the resolution of the currently used solution NMR techniques for probing such interactions. Future studies of antimicrobial peptide synergy will likely require stable isotope-labeling strategies, similar to those used in NMR studies of proteins.     

PII structure in the model peptides for unfolded proteins: studies on ubiquitin fragments and several alanine-rich peptides containing QQQ, SSS, FFF, and VVV

A great deal of attention has been paid lately to the structures in unfolded proteins due to the recent discovery of many biologically functional but natively unfolded proteins and the far-reaching implications of order in unfolded states for protein folding. Recently, studies on oligo-Ala, oligo-Lys, oligo-Asp, and oligo-Glu, as well as oligo-Pro, have indicated that the left-handed polyproline II (PII) is the major local structure in these short peptides. Here, we show by NMR and CD studies that ubiquitin fragments, model unfolded peptides composed of nonrepeating amino acids, and four alanine-rich peptides containing QQQ, SSS, FFF, and VVV sequences are all present in aqueous solution predominantly in the extended PII or beta conformation. The results from this and related studies indicate that PII might be a major backbone conformation in unfolded proteins. The presence of defined local backbone structure in unfolded proteins is inconsistent with predictions from random coil models.     

Review: Model Peptides and the Physicochemical Approach to β-Amyloids

β-Amyloid peptides are the main protein components of neuritic plaques and may be important in the pathogenesis of Alzheimer's Disease. The determination of the structure of β-amyloid fibrils poses a challenge because of the limited solubility of β-amyloid peptides and the noncrystalline nature of fibrils formed from these peptides. In this paper, we describe several physicochemical approaches which have been used to examine fibrils and the fibrillogenesis of peptide models of β-amyloid. Recent advances in solid state NMR, such as the DRAWS pulse sequence, have made this approach a particularly attractive one for peptides such as β-amyloid, which are not yet amenable to high-resolution solution phase NMR and crystallography. The application of solid state NMR techniques has yielded information on a model peptide comprising residues 10–35 of human β-amyloid and indicates that in fibrils, this peptide assumes a parallel β-strand conformation, with all residues in exact register. In addition, we discuss the use of block copolymers of Aβ peptides and polyethylene glycol as probes for the pathways of fibrillogenesis. These methods can be combined with other new methods, such as high-resolution synchrotron X-ray diffraction and small angle neutron and X-ray scattering, to yield structural data of relevance not only to disease, but to the broader question of protein folding and self-assembly.     

Conformation of sarafotoxin-6b in aqueous solution determined by NMR spectroscopy and distance geometry

The solution structure of sarafotoxin-6b in water has been determined using high-resolution NMR spectroscopy. 127 proton-proton distance measurements and three phi dihedral angle constraints derived from NMR spectra were used to calculate the solution structure using a combination of distance geometry and restrained molecular dynamics. The major structural feature of the resulting family of five structures was a right-handed alpha-helix extending from K9 to Q17. In contrast, the C-terminal region of the peptide appears not to adopt a preferred conformation in aqueous solution. The present structure is compared with those previously determined for endothelin peptides in non-aqueous solvents.     

Afc can adopt either the fully extended or a turn conformation

Summary We have synthesized by solution methods and fully characterized two sets of terminally protected peptides based on the tricyclic C^α,α-disubstituted glycine Afc. The conformational preferences of the Afc/Gly peptides were examined by FT-IR absorption and¹H NMR techniques, while those of the Afc/TOAC peptides were primarily investigated by using fluorescence spectroscopy. The X-ray diffraction structure of an Afc derivative was also analyzed. The body of solution and crystal-state experimental data conclusively confirms previous findings that the Afc residue may either adopt the fully extended (C₅) or a turn conformation.     

The distribution of alpha-helix propensity along the polypeptide chain is not conserved in proteins from the same family.

We address the question of whether the distribution of secondary structure propensities of the residues along the polypeptide chain (denominated here as secondary structure profiles) is conserved in proteins throughout evolution, for the particular case of alpha-helices. We have analyzed by CD the conformation of peptides corresponding to the five alpha-helices of two alpha/beta parallel proteins (ComA and Ara). The large alpha-helical population of peptide ComA-4 detected by CD in aqueous solution has been confirmed by NMR. These proteins are members of the CheY and P21-ras families, respectively, which have been studied previously in the same way (Muñoz V, Jiménez MA, Rico M, Serrano L, 1995, J Mol Biol 245:275-296). Comparison of the helical content of equivalent peptides reveals that protein alpha-helix propensity profiles are not conserved. Some equivalent peptides show very different helical populations in solution and this is especially evident in very divergent proteins (ComA and CheY). However, all the peptides analyzed so far adopted an important population of helical conformations in the presence of 30% trifluoroethanol, indicating that there could be a conserved minimal requirement for helical propensity.     

Stabilization of an alpha-helical conformation in an isolated hexapeptide inhibitor of calmodulin.

The conformational properties of two hexapeptides, Ac-LWRILW-NH(2) and its D-amino acid counterpart Ac-lwrilw-NH(2), identified as calmodulin inhibitors using mixture-based synthetic combinatorial library approaches, have been characterised by NMR and CD spectroscopy. The peptides fold into an alpha-helical conformation in aqueous solution. The observed short- and medium-range nuclear Overhauser effects were consistent with the formation of an alpha-helical structure and a reasonably well-defined set of structures was obtained by using restraints from the NMR data in simulated annealing calculations. Analysis of glycine-substitution analogues demonstrated that all the amino acids that make up the peptide sequence are important for the stabilization of the alpha-helical conformation. The results suggest that a well-defined set of interactions is indispensable to allow alpha-helix formation in this short hexapeptide.     

Comparison of the solution conformations of a cell-adhesive peptide LBE and its reverse sequence EBL

T-cell adhesion is mediated by an ICAM-1/LFA-1 interaction; this interaction plays a crucial role in T-cell activation during immune response. LBE peptide, which is derived from the beta-subunit of LFA-1, has been shown to inhibit ICAM-1/LFA-1-mediated T-cell adhesion. In this work, we studied the solution conformations of LBE peptide and its reverse sequence (EBL) by NMR, CD and molecular dynamics simulations. Reverse peptides have been used as controls in biological studies. The effect of reversing the sequence of LBE to EBL peptides on their respective conformations is important in understanding their biological properties in vitro or in vivo. The NMR studies for these peptides were carried out in water and in TFE/water solvent systems. In 40% TFE/water, both peptides exhibited helical conformation. CD studies suggested that the LBE exhibits 30% helical conformation, while the EBL exhibits 20% helical conformation. From the NMR and MD simulation studies, it was evident that the peptides exhibited a stable helical conformation; a stable helical structure was found at Leu6 to Leu15 for LBE and at Gly9 to Leu17 for EBL. The helical conformations of LBE and EBL may be in equilibrium with other possible conformers; the other conformers contain loop and turn structures. Both peptides bind to divalent cations because the LBE is derived from the cation-binding region of the LFA-1. This study shows that reversing the peptide sequence did not alter the secondary structure of the corresponding sequence. Hence, caution must be exercised when using reverse peptides as controls in biological studies. This report will improve our ability to design a better inhibitor of ICAM-1/LFA-1 interaction.     

Integrated analysis of the conformation of a protein-linked spin label by crystallography,EPR and NMR spectroscopy

Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein–protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.     

Methods of the site-selective solid phase synthesis of peptide-derived Amadori products

Two procedures of glycated peptides’ synthesis have been developed. The first method involves reductive alkylation of the ε-amino groups of lysine with 2,3:4,5-di-O-isopropylidene-β-d-arabino-hexos-2-ulo-2,6-pyranose in the presence of sodium cyanoborohydride on solid support. The second one uses a new fully protected lysine derivative, which is a building block designed for direct introduction of the glycated lysine moiety into a peptide, according to the standard solid phase synthesis protocol. The applicability of the proposed methods for the synthesis of peptide-derived Amadori products is discussed. The structure of the synthesized glycated peptides was confirmed by high-resolution mass spectrometry and enzymatic hydrolysis. Circular dichroism studies, performed in water solution, revealed that the formation of the Amadori rearrangement product in the lysine side chain does not influence significantly the conformational preferences of the peptides studied. However, when the solvent was changed to trifluoroethanol, the glycated peptides preferred β-turn conformation.     

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH

Structure determination of proteins by NMR is unique in its ability to measure restraints, very accurately, in environments and under conditions that closely mimic those encountered in vivo. For example, advances in solid-state NMR methods enable structure determination of membrane proteins in detergent-free lipid bilayers, and of large soluble proteins prepared by sedimentation, while parallel advances in solution NMR methods and optimization of detergent-free lipid nanodiscs are rapidly pushing the envelope of the size limit for both soluble and membrane proteins. These experimental advantages, however, are partially squandered during structure calculation, because the commonly used force fields are purely repulsive and neglect solvation, Van der Waals forces and electrostatic energy. Here we describe a new force field, and updated energy functions, for protein structure calculations with EEFx implicit solvation, electrostatics, and Van der Waals Lennard-Jones forces, in the widely used program Xplor-NIH. The new force field is based primarily on CHARMM22, facilitating calculations with a wider range of biomolecules. The new EEFx energy function has been rewritten to enable OpenMP parallelism, and optimized to enhance computation efficiency. It implements solvation, electrostatics, and Van der Waals energy terms together, thus ensuring more consistent and efficient computation of the complete nonbonded energy lists. Updates in the related python module allow detailed analysis of the interaction energies and associated parameters. The new force field and energy function work with both soluble proteins and membrane proteins, including those with cofactors or engineered tags, and are very effective in situations where there are sparse experimental restraints. Results obtained for NMR-restrained calculations with a set of five soluble proteins and five membrane proteins show that structures calculated with EEFx have significant improvements in accuracy, precision, and conformation, and that structure refinement can be obtained by short relaxation with EEFx to obtain improvements in these key metrics. These developments broaden the range of biomolecular structures that can be calculated with high fidelity from NMR restraints.