Solution Structure Of Peptide Research Articles

Combination of circular dichroism spectroscopy and size-exclusion chromatography coupled with HDX-MS for studying global conformational structures of peptides in solution

Misfolding of therapeutic peptides and proteins can lead to numerous issues, ranging in severity, including loss of function, aggregation, immunogenicity, and cytotoxicity. A primary component of protein folding is secondary structure, including α-helices and β-sheets. Many native peptides and proteins are predominately α-helical therefore, it is of critical importance to develop robust and reliable analytical tools to investigate protein higher order structure, including the percentage of α-helix under various conditions, to evaluate protein folding and prevent the negative effects of misfolding. However, given the complexity of protein folding and higher order structure, it is unlikely that one technique will provide a comprehensive analysis. To bridge this gap, this study presents the combination of two orthogonal techniques – circular dichroism (CD) and size-exclusion chromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS) to investigate global peptide and protein conformations. Also, the incorporation of trifluoroethanol (TFE), a known stabilizer of α-helical structures, into the analyses, aims to enhance the discrimination power of these two techniques by increasing the alpha helical stability range of study. CD data was used to estimate the percent of α-helix content and its thermal stability while online SEC-HDX-MS screening compared global conformational changes of each peptide based on a difference in the number of deuterons exchanged to protons, ΔHDX. The workflow described in this report can be very beneficial in pharmaceutical development. The model peptides were chosen to demonstrate the workflow with commercially available compounds. The goal of this study was to show a proof-of-concept for direct correlation of these methodologies and to estimate the percentage of α-helix content at a particular ΔHDX, which is indicative of the state of protein folding.

Solution Study of Engineered Quartz Binding Peptides Using Replica Exchange Molecular Dynamics

We use replica-exchange molecular dynamics (REMD) to interrogate molecular structures and properties of four engineered dodecapeptides (in solution, in the absence of a surface) that have been shown to bind to quartz with different propensities. We find that all of the strong-binding peptides feature some polyproline type II secondary structure, have less conformational freedom, and feature fewer intrapeptide hydrogen bonds compared with the weak binder. The regions of contiguous proline content in a given sequence appear to play a role in fostering some of these properties of the strong binders. For preliminary insights into quartz binding, we perform lattice-matching studies between a grid corresponding with the quartz (100) surface and the strong-binding peptide REMD structures. Our findings indicate a commonality among the putative contact residues, even for peptide structures with very different backbone conformations. Furthermore, interpeptide interactions in solution are studied. Our preliminary findings indicate that the strong-binder interpeptide contacts are dominated by weak, nonspecific hydrophobic interactions, while the weak-binding peptide shows more variable behavior due to the distribution of charged residues. In summary, the solution structures of peptides appear to be significant. We propose that these differences in their intra- and interpeptide interactions can influence their propensity to bind onto a solid substrate.

Combination of SAXS and NMR Techniques as a Tool for the Determination of Peptide Structure in Solution

The biological activity of peptides is largely determined by their spatial structure in solution. Because of flexibility of these molecules, the NMR technique, commonly used to determine the solution structures, usually provides only the information of the local geometry of peptide chains. On the other hand, small-angle X-ray scattering (SAXS) gives interatomic distance distribution in the molecule, thereby defining the shape of a molecule. In this work we carried out a SAXS study of the 28−43 sequence fragment of the B3 domain of immunoglobulin binding protein G from Streptococcus, IG(28−43). We fitted the conformational ensembles obtained in our previous work by using the NMR-restrained molecular dynamics simulations to the distance distribution obtained from SAXS measurements. Only two clusters of conformations with shape bent in the middle out of 11, consistent with NMR data had nonzero statistical weights. Thus, SAXS and NMR provide complementary structural information and appear to be an excellent t...

Structure Analysis of Activity-dependent Neurotrophic Factor 9 by Circular Dichroism and Sedimentation Equilibrium

Activity-dependent neurotrophic factor (ADNF) is a glia-derived neurotrophic peptide, which protects neurons from tetrodoxin treatment and Alzheimer's disease-related and amyotrophic-lateral-sclerosis-related insults at femto-molar concentrations. However, the mechanism of the femto-molar neuroprotection by the peptide has not been elucidated. The characterization of the peptide structure in solution at molecular level should shed light in the mechanism of such extremely high biological activity. From that point of view, the secondary and quaternary structure analysis of ADNF9, an active core fragment peptide of ADNF, was performed by circular dichroism (CD) and sedimentation equilibrium. ADNF9 has also been shown to exhibit a neurotrophic activity in femto-molar concentrations; in this study it showed sub-pM neuroprotective activity against V642I-APP-induced cytotoxity in the mouse primary cortical neuron. CD analysis showed that the secondary structure of ADNF9 is identical in water and phosphate-buffered saline (PBS) and is independent of the peptide concentration. The CD spectra appear to be characterized most likely as disordered. The sedimentation equilibrium experiments demonstrated monomeric structure of the protein over the wide range of peptide concentration. There is a slight enhancement of CD intensity at 37 degrees C relative to 20 degrees C, suggesting a possible hydrophobic association of the peptide. There is no change in the secondary structure in PBS upon freeze-thaw treatment, which has previously been suggested to cause activity loss.

The search for molecular determinants of LPS inhibition by proteins and peptides.

Lipo-poly-saccharide (LPS) induced Gram-negative sepsis and septic shock remain lethal in up to 60 % of cases, and LPS antagonists that neutralize its endotoxic action are the subject of intensive research. The molecular motifs of specific binding of LPS by antiendotoxin proteins and peptides may lead to an understanding of LPS action at the atomic level and provide clues for the development of new immunomodulatory compounds for use as therapy in the treatment of Gram-negative bacterial sepsis. The interaction of LPS with its cognate binding proteins has been structurally elucidated in the single case of the X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from E. coli K-12 (Ferguson et al., Science 1999, 282, 2215). This structure and other known structures of LPS binding proteins have been used to propose a common binding motif of LPS to proteins. Another independent source of structural information are solution structures of peptides in complex with LPS that can be determined using the transferred NOE effect. The molecular mechanisms of biological activity of bacterial endotoxins can additionally be probed by theoretical means. The growing structural knowledge is opening pathways to the design of peptides or peptidomimetics with improved antiendotoxin properties.

The Role of Hydrophobic Interactions in Amyloidogenesis: Example of Prion-Related Polypeptides

Conversion of the non-infectious, cellular forom of the prion protein (PrPC) to the infectious form (PrPSc) is thought to be driven by an α-helical to β-sheet conformational transition. To reveal the sequence determinants which encourage the transition to (β-fold, we study the synthetic peptides associated with hydrophobic conserved fragments of the N-terminal region of the prion protein. The structure of peptides in solution was probed under various thermodynamic conditions employing circular dichroism and sleady stale fluorescence spectroscopy as well as dye binding assays. The fluorescence methods utilized pyrene residues covalently attached to the end of the model peptides. In aqueous solutions, the structure assessments indicate the formation of metastable peptide aggregates; the molecular conformations within the peptide micelles are largely coiled. This stage in molecular assembly exists without significant β-strand formation, i.e., before the appearance of any ordered secondary structure delectable by circular dichroism. At moderate concentrations of trifluo-roethanol and/or acetonitrile, the conformational ensemble shifts towards β-strand formation, and the population of the amorphous aggregates decreases significantly. Overall, the present data indicate that hydrophobic interactions between side chains of the peptide variants prevent, in fact, the formation of the rigid (β-sheet structures. Encouragement of β-folds requires the deslabilizalion of local interactions in the peptide chain, which in vivo might be possible within cell membranes as well as within partly folded molecular forms

Structural characterization of peptide fragments from hCD81-LEL.

The solution conformation of two peptides [1: PSGSNIISNLFKED; 2: GSSTLTALTTSVLKNNL] from human CD81 (hCD81) large extra-cellular loop (LEL) with known importance in the hepatitis C virus glycoprotein E2 (HCV-E2) binding interaction was characterized using circular dichroism spectroscopy. In addition, the solution structure of peptide 1 that contains a phenylalanine residue (F186 in hCD81) known to be critical in the binding interaction with HCV-E2 was determined using 1D and 2D 1H NMR spectroscopy. Both peptides are unstructured in water but begin forming significant helical conformation following the addition of 20% or more trifluoroethanol (v/v), a result consistent with their alpha-helical conformation found in the native protein. The CD data recorded as a function of pH and NaCl concentration are consistent with stabilization of the helical structure from electrostatic forces for both peptides. Peptide 1 is able to block the binding interaction of recombinant HCV-E2 (rHCV-E2) to hCD81 expressed on Molt-4 T cells at high concentrations (3.5 mM), a low affinity that we attributed to the random coil structure in water.

Tripeptides adopt stable structures in water. A combined polarized visible Raman, FTIR, and VCD spectroscopy study.

We have measured the band profile of amide I in the infrared, isotropic, and anisotropic Raman spectra of L-alanyl-D-alanyl-L-alanine, acetyl-L-alanyl-L-alanine, L-vanyl-L-vanyl-L-valine, L-seryl-L-seryl-L-serine, and L-lysyl-L-lysyl-L-lysine at acid, neutral, and alkaline pD. The respective intensity ratios of the two amide I bands depend on the excitonic coupling between the amide I modes of the peptide group. These intensity ratios were obtained from a self-consistent spectral decomposition and then were used to determine the dihedral angles between the two peptide groups by means of a recently developed algorithm (Schweitzer-Stenner, R. Biophys. J. 2002, 83, 523-532). The validity of the obtained structures were checked by measuring and analyzing the vibrational circular dichroism of the two amide I bands. Thus, we found two solutions for all protonation states of trialanine. Assuming a single conformer, one obtains a very extended beta-helix-like structure. Alternatively, the data can be explained by the coexistence of a 3(1)(PII) and a beta-sheet-like structure. Acetyl-L-alanyl-L-alanine exhibits a structure which is very similar to that obtained for trialanine. The tripeptide with the central D-alanine adopts an extended structure with a negative psi and a positive phi angle. Trivaline and triserine adopt single beta(2)-like structures such as that identified in the energy landscape of the alanine dipeptide. Trilysine appears different from the other investigated homopeptides in that it adopts a left-handed helix which at acid pD is in part stabilized by hydrogen bonding between the protonated carboxylate (donor) and the N-terminal peptide carbonyl. Our result provides compelling evidence for the capability of short peptides to adopt stable structures in an aqueous solution, which at least to some extent reflect the intrinsic structural propensity of the respective amino acids in proteins. Furthermore, this paper convincingly demonstrates that the combination of different vibrational spectroscopies provides a powerful tool for the determination of the secondary structure of peptides in solution.

Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest

Vpr, one of the accessory gene products encoded by HIV-1, is a 96-residue protein with a number of functions, including targeting of the viral pre-integration complex to the nucleus and inducing growth arrest of dividing cells. We have characterized by 2D NMR the solution conformations of bioactive synthetic peptide fragments of Vpr encompassing a pair of H(F/S)RIG sequence motifs (residues 71-75 and 78-82 of HIV-1 Vpr) that cause cell membrane permeabilization and death in yeast and mammalian cells. Due to limited solubility of the peptides in water, their structures were studied in aqueous trifluoroethanol. Peptide Vpr59-86 (residues 59-86 of Vpr) formed an alpha-helix encompassing residues 60-77, with a kink in the vicinity of residue 62. The first of the repeated sequence motifs (HFRIG) participated in the well-defined alpha-helical domain whereas the second (HSRIG) lay outside the helical domain and formed a reverse turn followed by a less ordered region. On the other hand, peptides Vpr71-82 and Vpr71-96, in which the sequence motifs were located at the N-terminus, were largely unstructured under similar conditions, as judged by their C(alpha)H chemical shifts. Thus, the HFRIG and HSRIG motifs adopt alpha-helical and turn structures, respectively, when preceded by a helical structure, but are largely unstructured in isolation. The implications of these findings for interpretation of the structure-function relationships of synthetic peptides containing these motifs are discussed.

Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest.

Vpr, one of the accessory gene products encoded by HIV-1, is a 96-residue protein with a number of functions, including targeting of the viral pre-integration complex to the nucleus and inducing growth arrest of dividing cells. We have characterized by 2D NMR the solution conformations of bioactive synthetic peptide fragments of Vpr encompassing a pair of H(F/S)RIG sequence motifs (residues 71-75 and 78-82 of HIV-1 Vpr) that cause cell membrane permeabilization and death in yeast and mammalian cells. Due to limited solubility of the peptides in water, their structures were studied in aqueous trifluoroethanol. Peptide Vpr59-86 (residues 59-86 of Vpr) formed an alpha-helix encompassing residues 60-77, with a kink in the vicinity of residue 62. The first of the repeated sequence motifs (HFRIG) participated in the well-defined alpha-helical domain whereas the second (HSRIG) lay outside the helical domain and formed a reverse turn followed by a less ordered region. On the other hand, peptides Vpr71-82 and Vpr71-96, in which the sequence motifs were located at the N-terminus, were largely unstructured under similar conditions, as judged by their C(alpha)H chemical shifts. Thus, the HFRIG and HSRIG motifs adopt alpha-helical and turn structures, respectively, when preceded by a helical structure, but are largely unstructured in isolation. The implications of these findings for interpretation of the structure-function relationships of synthetic peptides containing these motifs are discussed.

Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest

Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest

Infrared evidence of a β-hairpin peptide structure in solution

The IR spectrum of an 16-amino acid peptide corresponding, according to NMR studies, to a β-hairpin has been analysed. Two characteristic features distinguish its spectrum from that of an antiparallel β-sheet: the low-frequency band that in a β-sheet structure is located at 2 ∼ 1632 cm −1 appears here at 2∼ 1620 cm −1, and the high-frequency component does not undergo the isotopic shift typical of β-sheet from 1690 to 1675 cm −1 when transferred to D 2O. The infrared characteristics associated with β-hairpins have been described so far in two proteins, in one of which, whose three-dimensional structure is known from X-ray diffraction, a β-hairpin has actually been detected.

De novo design and structural characterization of an alpha-helical hairpin peptide: a model system for the study of protein folding intermediates.

The de novo design and structural characterization of an alpha-helical hairpin peptide (alpha-helix/turn/alpha-helix, alpha t alpha) are reported. The peptide is intended to provide a model system for the study of the interactions of secondary structural elements during protein folding. Both the diffusion-collision and framework models of protein folding envision that the earliest intermediates in protein folding are transient secondary structures or microdomains which interact and become mutually stabilizing. Design principles for the alpha t alpha peptide were drawn from the large body of work on the structure of peptides in solution. Computer modeling was not used in the design process. Study of alpha t alpha by circular dichroism and two-dimensional nuclear magnetic resonance indicates that the designed peptide is monomeric, helical, and stable in aqueous solution at room temperature. Analysis of two-dimensional nuclear magnetic resonance experiments indicates that the two helices and the turn form in the intended positions and that the helices associate in the designed orientation. Development of alpha t alpha represents an advance in protein design in that both the secondary structural elements and designed tertiary interactions have been realized and can be detected in solution by nuclear magnetic resonance. The resulting model system resembles a protein folding intermediate and will allow the study of interacting helices in a context that approximates an early stage in protein folding.

A1H Paramagnetic Relaxation Study on the Interaction of Peptides with Aminoxyl Spin Labels in Apolar Environments

Abstract The use of stable free radicals such as aminoxyl spin-labels has been recently exploited for obtaining information on solution structures of peptides (1,2) and proteins (3,4) from multidimensional NMR spectra. In those reports, it has been suggested that an efficient spin-labelling of water and dimethylsulphoxide (DMSO) solvents may be achieved and the paramagnetic solvent yields relaxation effects on backbone proton nuclei which are directly driven by the molecular surface accessibility. Upon the addition of the paramagnetic probe, the observation of absence in chemical shifts changes and of nuclear relaxation effects which are fully rationalised in terms of the solution structure, support a dynamic model for the interaction between the chemical probe and the biomolecule where weak collisional adducts favour the dipolar coupling of the unpaired electron with the outer nuclei of the investigated molecule.

Solution structure of peptides containing two dehydro‐phenylalanine residues: A CD investigation

The peptides Ac-delta Phe-Ala-delta Phe-NH-Me (1), Ac-delta Phe-Val-delta Phe-NH-Me (2), Ac-delta Phe-Gly-delta Phe-Ala-OMe (3), and Boc-Ala-delta Phe-Gly-delta Phe-Ala-OMe (4), containing two dehydro-phenylalanine (delta Phe) residues, were synthesized and the solution structure investigated in various solvents. The nmr and CD measurements indicate that all the dehydropeptides examined adopt 3(10)-helical conformations in solution. The tripeptides 1 and 2 exhibited an intense negative CD exciton couplet, which was assigned to the right-handed screw sense, while the tetrapeptide 3 displayed a CD couplet having opposite sign, which was assigned to the left-handed helical sense. In the pentapeptide 4 the sense of the helix was found to vary with solvent and temperature, as demonstrated by the sign reversal of the CD spectrum. The right-handed sense dominates in hexafluoro-2-propanol, whereas a left-handed helix prevails in chloroform, acetonitrile and methanol. A crucial role for this behavior is likely to be played by the two alanine residues positioned respectively at the head and tail of the sequence, which favor conformations having opposite screw senses.

Vasoactive intestinal contractor (VIC) peptide structure in solution as determinated by proton NMR

The Vasoactive Intestinal Contractor peptide has been synthesized and studied by 1 H NMR in 50/50 acetonitrile/water mixture. All spin systems were identified and assigned with the aid of 2D experiments. Modeling was performed with constraints obtained by NOEs. Some distances used in modeling were calculated from build-up data. Kinetic exchange of the amide protons showed that the 9-15 helical segment is stabilized by at least three hydrogen bonds. The proposed structure is compared with those of Nle-7 endothelin and Sarafotoxin-S6b previously obtained in same conditions.

Some aspects of NMR techniques for the conformational analysis of peptides

An outline is given of some new NMR spectroscopic approaches to the spatial structure of peptides in solution which have been recently suggested and tested in the Shemyakin Institute for Chemistry of Natural Products. The previously derived angular dependence of the peptide vicinal 3'NHCH coupling constant has been refined on the basis of the latest experimental data. It has been found that ion—dipole interaction of the type C==-O M +(where M is an alkali metal ion) leads to a low field shift of the '3C signal of the carbonyl group. This effect permits determination of the number and location of the ligand groups which form the internal cavity of peptide and depsipeptide complexones. The effect of 'shift reagents' on the NMR spectra sheds certain light on the spatial structure of peptides in solution, and, in particular, gives considerable information on the configuration of the amide bond and on the rotational states of the C—-C bond. The INDOR and signals on combination frequency have been used for detection of 'hidden' signals (their multiplicity, chemical shift and splitting) and on the assignment of the NH signals in the proton NMR spectra of peptides. High resolution NMR spectroscopy is becoming the most powerful means for the study of the spatial structure of peptides in solution. The progress in this field has required the development of new spectroscopic approaches. The subject matter of this paper is to outline some of those which have been suggested and tested recently in the Shemyakin Institute for Chemistry of Natural Products. I. THE ANGULAR DEPENDENCE OF THE PEPTIDE VICINAL NH-C8H COUPLING CONSTANT With the accumulation of more experimental data it has now become possible to refine the earlier proposed2 dependence of the 3NflCH constant on the dihedral angle U between the H—N---C' and N—C-----H planes.