Three-dimensional structure of gurmarin,a sweet taste-suppressing polypeptide

Summary The solution structure of gurmarin was studied by two-dimensional proton NMR spectroscopy at 600 MHz. Gurmarin, a 35-amino acid residue polypeptide recently discovered in an Indian-originated tree Gymnema sylvestre, selectively suppresses the neural responses of rat to sweet taste stimuli. Sequence-specific protons. The three-dimensional solution structure was determined by simulated-annealing calculations on the basis of 135 interproton distance constraints derived from NOEs, six distance constraints for three hydrogen bonds and 16 dihedral angle constraints derived from coupling constants. A total of 10 structures folded into a well-defined structure with a triple-stranded antiparallel -sheet. The average rmsd values between any two structures were 1.65±0.39 Å for the backbone atoms (N, C, C) and 2.95±0.27 Å for all heavy atoms. The positions of the three disulfide bridges, which could not be deterermined chemically, were estimated to be Cys³–Cys¹⁸, Cys¹⁰–Cys²³ and Cys¹⁷–Cys³³ on the basis of the NMR distance constraints. This disulfide bridge pattern in gurmarin turned out to be analogous to that in -conotoxin and Momordica charantia trypsin inhibitor-II, and the topology of folding was the same as that in -conotoxin.Abbreviations DQF-COSY double-quantum-filtered correlated spectroscopy - HOHAHA homonuclear Hartmann-Hahn spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy - ppm parts per million; rmsd, root-mean-square deviation - TSP 3-(trimethylsilyl)-2,2,3,3-tetradeutero-propionate     

Complete relaxation matrix refinement of NMR structures of proteins using analytically calculated dihedral angle derivatives of NOE intensities

Summary A new method for refining three-dimensional (3D) NMR structures of proteins is described, which takes account of the complete relaxation pathways. Derivatives of the NOE intensities with respect to the dihedral angles are analytically calculated, and efficiently evaluated with the use of a filter technique for identifying the dominant terms of these derivatives. This new method was implemented in the distance geometry program DIANA. As an initial test, we refined 30 rigid distorted helical structures, using a simulated data set of NOE distance constraints for a rigid standard -helix. The final root-mean-square deviations of the refined structures relative to the standard helix were less than 0.1 Å, and the R-factors dropped from values between 7% and 32% to values of less than 0.5% in all cases, which compares favorably with the results from distance geometry calculations. In particular, because spin diffusion was not explicitly considered in the evaluation of exact¹H–¹H distances corresponding to the simulated NOE intensities, a group of nearly identical distance geometry structures was obtained which had about 0.5 Å root-mean-square deviation from the standard -helix. Further test calculations using an experimental NOE data set recorded for the protein trypsin inhibitor K showed that the complete relaxation matrix refinement procedure in the DIANA program is functional also with systems of practical interest.Abbreviations RMSD root-mean-square deviation - NOE nuclear Overhauser enhancement - NOESY 2-dimensional nuclear Overhauser enhancement spectroscopy - CPU central processing unit     

Validation of the use of intermolecular NOE constraints for obtaining docked structures of protein-ligand complexes

Summary The use of intermolecular NOEs for docking a small ligand molecule into its target protein has been investigated with the aim of determining the effectiveness and methodology of this type of NOE docking calculation. A high-resolution X-ray structure of a protein-ligand complex has been used to simulate loose distance constraints of varying degrees of quality, typical of those estimated from experimental NOE intensities. These simulated data were used to examine the effect of the number, distribution and representation of the experimental constraints on the precision and accuracy of the calculated structures. A standard simulated annealing protocol was used, as well as a more novel method based on rigid-body dynamics. The results showed some analogies with those from similar studies on complete protein NMR structure determinations, but it was found that more constraints per torsion angle are required to define docked structures of similar quality. The effectiveness of different NOE-constraint averaging methods was explored and the benefits of using R^–6 averaging rather than centre averaging with small sets of NOE constraints were shown. The starting protein structure used in docking calculations was obtained from previous X-ray or NMR structure studies on a related complex. The effects on the calculated conformations of introducing structural differences into the binding site of the initial protein structure were also considered.To whom correspondence should be addressed.     

Synthesis of a bicyclic BPTI mimetic containing 4-thioproline replacing Cys38

The synthesis of a backbone bicyclic nonapeptide that mimics the binding site of bovine pancreatic trypsin inhibitor (BPTI) is described. The BPTI mimetic, which contains cis-thioproline replacing Cys³⁸ of the protein, inhibits trypsin with a K_i of 76 M.     

High-resolution solution structure of siamycin II: Novel amphipathic character of a 21-residue peptide that inhibits HIV fusion

Summary The 21-amino acid peptides siamycin II (BMY-29303) and siamycin I (BMY-29304), derived from Streptomyces strains AA3891 and AA6532, respectively, have been found to inhibit HIV-1 fusion and viral replication in cell culture. The primary sequence of siamycin II is CLGIGSCNDFAGCGYAIVCFW. Siamycin I differs by only one amino acid; it has a valine residue at position 4. In both peptides, disulfide bonds link Cys¹ with Cys¹³ and Cys⁷ with Cys¹⁹, and the side chain of Asp⁹ forms an amide bond with the N-terminus. Siamycin II, when dissolved in a 50:50 mixture of DMSO and H₂O, yields NOESY spectra with exceptional numbers of cross peaks for a peptide of this size. We have used 335 NOE distance constraints and 13 dihedral angle constraints to generate an ensemble of 30 siamycin II structures; these have average backbone atom and all heavy atom rmsd values to the mean coordinates of 0.24 and 0.52 Å, respectively. The peptide displays an unusual wedge-shaped structure, with one face being predominantly hydrophobic and the other being predominantly hydrophilic. Chemical shift and NOE data show that the siamycin I structure is essentially identical to siamycin II. These peptides may act by preventing oligomerization of the HIV transmembrane glycoprotein gp41, or by interfering with interactions between gp41 and the envelope glycoprotein gp120, the cell membrane or membrane-bound proteins [Frèchet, D. et al. (1994) Biochemistry, 33, 42–50]. The amphipathic nature of siamycin II and siamycin I suggests that a polar (or apolar) site on the target protein may be masked by the apolar (or polar) face of the peptide upon peptide/protein complexation.Abbreviations ABNR adopted basis Newton Raphson - AIDS acquired immunodeficiency syndrome - CW continuous wave - DMSO dimethylsulfoxide - DQF-COSY two-dimensional double-quantum-filtered correlation spectroscopy - HIV human immunodeficiency virus - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - ppm parts per million - P.E.-COSY two-dimensional primitive exclusive correlation spectroscopy - REDAC redundant dihedral angle constraint - rf radio frequency - rmsd root-mean-square difference - SIV simian immunodeficiency virus - sw spectral width - _m mixing time - TOCSY two-dimensional total correlation spectroscopy - TSP trimethylsilyl-2,2,3,3-²H₄-propionate - 2D two-dimensional     

Intrinsic nature of the three-dimensional structure of proteins as determined by distance geometry with good sampling properties

Summary A protocol for distance geometry calculation is shown to have excellent sampling properties in the determination of three-dimensional structures of proteins from nuclear magnetic resonance (NMR) data. This protocol uses a simulated annealing optimization employing mass-weighted molecular dynamics in four-dimensional space (Havel, T.F. (1991) Prog. Biophys. Mol. Biol., 56, 43–78). It attains an extremely large radius of convergence, allowing a random coil conformation to be used as the initial estimate for the succeeding optimization process. Computations are performed with four systems of simulated distance data as tests of the protocol, using an unconstrained l-alanine 30mer and three different types of proteins, bovine pancreatic trypsin inhibitor, the -amylase inhibitor Tendamistat, and the N-terminal domain of the 434-repressor. The test of the unconstrained polypeptide confirms that the sampled conformational space is that of the statistical random coil. In the larger and more complicated systems of the three proteins, the protocol gives complete convergence of the optimization without any trace of initial structure dependence. As a result of an exhaustive conformational sampling by the protocol, the intrinsic nature of the structures generated with distance restraints derived from NMR data has been revealed. When the sampled structures are compared with the corresponding X-ray structures, we find that the averages of the sampled structures always show a certain pattern of discrepancy from the X-ray structure. This discrepancy is due to the short distance nature of the distance restraints, and correlates with the characteristic shape of the protein molecule.Abbreviations r.m.s.d. root-mean-square deviation - MD molecular dynamics - NMR nuclear magnetic resonance - NOE nuclear Overhauser enhancement - BPTI bovine pancreatic trypsin inhibitor     

3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

Summary A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of¹H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the and backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His¹¹⁹ side chain in the solution and crystal structures has been detected.     

Three-dimensional structure of echistatin and dynamics of the active site

Summary The snake venom protein echistatin contains the cell recognition sequence Arg-Gly-Asp and is a potent inhibitor of platelet aggregation. The three-dimensional structure of echistatin and the dynamics of the active RGD site are presented. A set of structures was determined using the Distance Geometry method and subsequently refined by Molecular Dynamics and energy minimization. Disulfide pairings are suggested, based on violations of experimental constraints. The structures satisfy 230 interresidue distance constraints, derived from nuclear Overhauser effect measurements, five hydrogen-bonding constraints, and 21 torsional constraints from vicinal spin-spin coupling constants. The segment from Gly⁵ to Cys²⁰ and from Asp³⁰ to Asn⁴² has a well-defined conformation and the Arg-Gly-Asp sequence, which adopts a turn-like structure, is located at the apex of a nine-residue loop connecting the two strands of a distorted -sheet. The mobility of the Arg-Gly-Asp site has been quantitatively characterized by ¹⁵N relaxation measurements. The overall correlation time of echistatin was determined from fluorescence measurements, and was used in a model-free analysis to determine internal motional parameters. The active site has order parameters of 0.3–0.5, i.e., among the smallest values ever observed at the active site of a protein. Correlation of the flexible region of the protein as characterized by relaxation experiments and the NMR solution structures was made by calculating generalized order parameters from the ensemble of three-dimensional structures. The motion of the RGD site detected experimentally is more extensive than a simple RGD loop wagging motional model, suggested by an examination of superposed solution structures.     

Structural and dynamic studies of two antigenic loops from haemagglutinin: A relaxation matrix approach

Summary We have investigated the dynamics and structural behaviour of two antigenic peptides using ¹H NMR. The two cyclic peptides mimic the antigenic site A of influenza haemagglutinin protein; they only differ in the way they were cyclized and in the size of their respective linkers. Homonuclear relaxation parameters extracted from a complete NOE matrix were interpreted in terms of local dynamics. A set of distance constraints was deduced from these parameters which allowed 3D models to be constructed using distance geometry. NOE back-calculation was used to check the validity of the final models. Strong variations of internal motion amplitude have been found in both peptides along their backbone. Motions with high amplitudes have been localized in the Gly-Pro-Gly sequence which forms a -turn in both structures.Abbreviations DSS 3-(trimethylsilyl)-1-propanesulfonic acid - D-loop aspartic acid loop - ELISA enzyme-linked immunoabsorbent assay - f.i.d free induction decay - HOHAHA homonuclear Hartmann-Hahn spectroscopy - HPLC high pressure liquid chromatography - K-loop lysine loop - NMR nuclear magnetic resonance - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - r.m.s.d. root-mean-square deviation of atomic positions     

Treatment of NOE constraints involving equivalent or nonstereoassigned protons in calculations of biomacromolecular structures

Summary Two modifications to the commonly used protocols for calculating NMR structures are developed, relating to the treatment of NOE constraints involving groups of equivalent protons or nonstereoassigned diastereotopic protons. Firstly, a modified method is investigated for correcting for multiplicity, which is applicable whenever all NOE intensities are calibrated as a single set and categorised in broad intensity ranges. Secondly, a new set of values for pseudoatom corrections is proposed for use with calculations employing centre-averaging. The effect of these protocols on structure calculations is demonstrated using two proteins, one of which is well defined by the NOE data, the other less so. It is shown that failure to correct for multiplicity when using r^-6 averaging results in overly precise structures, higher NOE energies and deviations from geometric ideality, while failure to correct for multiplicity when using r^-6 summation can cause an avoidable degradation of precision if the NOE data are sparse. Conversely, when multiplicities are treated correctly, r^-6 averaging, r^-6 summation and centre averaging all give closely comparable results when the structure is well defined by the data. When the NOE data contain less information, r^-6 averaging or r^-6 summation offer a significant advantage over centre averaging, both in terms of precision and in terms of the proportion of calculations that converge on a consisten result.Abbreviations HMG high mobility group - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - rmsd root-mean-square deviation - YASAP yet another simulated-annealing protocol     

Protein dynamics studied by rotating frame 15N spin relaxation times

Summary Conformational rate processes in aqueous solutions of uniformly ¹⁵N-labeled pancreatic trypsin inhibitor (BPTI) at 36°C were investigated by measuring the rotating frame relaxation times of the backbone ¹⁵N spins as a function of the spin-lock power. Two different intramolecular exchange processes were identified. A first local rate process involved the residues Cys³⁸ and Arg³⁹, had a correlation time of about 1.3 ms, and was related to isomerization of the chirality of the disulfide bond Cys¹⁴-Cys³⁸. A second, faster motional mode was superimposed on the disulfide bond isomerization and was tentatively attributed to local segmental motions in the polypeptide sequence-Cys¹⁴-Ala¹⁵-Lys¹⁶-. The correlation time for the overall rotational tumbling of the protein was found to be 2 ns, using the assumption that relaxation is dominated by dipolar coupling and chemical shift anistropy modulated by isotropic molecular reorientation.Abbreviations BPTI basic pancreatic trypsin inhibitor - 2D two-dimensional - COSY 2D correlation spectroscopy - TOCSY 2D total correlation spectroscopy - RF radio frequency - CW continuous wave - TPPI time-proportional phase incrementation - CSA chemical shift anisotropy - T₁ longitudinal relaxation time - T₂ transverse relaxation time - T₁ relaxation time in the rotating frame , correlation time for overall rotational reorientation of the protein - _ex ^s , _ex ^f , correlation times for two conformational exchange processes (slow and fast).     

Distance-constraint approach to protein folding. II. Prediction of three-dimensional structure of bovine pancreatic trypsin inhibitor

The possibility of predicting the three-dimensional structure of bovine pancreatic trypsin inhibitor (BPTI) is examined using a distance-constraint approach. The mean distances between the amino acid residues in globular proteins, calculated in a previous paper, are utilized as distance constraints. In this study, as in previous work of others, root-mean-square deviations of the predicted conformations from the native one of less than 4 Å could not be obtained if the only input information consisted of the mean distances between amino acid residues (which involve information about the hydrophobicity and hydrophilicity of each amino acid residue) and the locations of disuifide bonds, -helices, and -structures. An examination is made of the kinds of structural features of BPTI that appear in the conformations predicted without explicit inclusion of information about such structural features, and of the kinds of information required in a given set of distance constraints for successful folding of BPTI. For example, structures that resemble incipient-forming -helices, bends, and -structures are observed in the conformations predicted when only the mean distances between the amino acid residues and the locations of the disulfide bonds (without information about the locations of -helices, bends, and -structures) are used. One type of additional required information is knowledge of spatially distant pairs involving the active site and the terminal residues. Furthermore, examination of the missing information indicates an important role for the strong nonbonded interaction between sulfur atoms and the side chains of aromatic amino acid residues in BPTI. When such information is introduced into the set of distance constraints, in terms of the exact distances of five pairs, (which implicitly include sulfur/aromatic interactions), the root-mean-square deviations of the predicted conformations decrease to 2.2–3.2 Å. Several methods for comparing conformations are also discussed; in particular, comparisons between conformations of short segments are carried out by a differential-geometry procedure.     

A refined three-dimensional solution structure of a carboxy terminal fragment of apolipoprotein CII

The three-dimensional structure of a synthetic fragment of human apolipoprotein CII (apo-CII) in 35%, 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) has been determined on the basis of distance and intensity constraints derived from two-dimensional proton nuclear magnetic resonance measurements. The NOE crosspeak build-up rates were converted to distance constraints which were used in the distance geometry program DIANA. A set of one hundred structures were generated and of these ten structures were used in molecular dynamics simulations using the program XPLOR. This program enabled a direct minimization between the difference of the two-dimensional NOE intensities and those calculated from the full relaxation matrix. In this way spin diffusion is fully taken into account, which can be seen from the considerable improvement of the R-factor after the relaxation matrix refinement. These calculations show that this fragment, which corresponds to the carboxy terminal 30 amino acids of intact apo-CII and which retains its ability to activate lipoprotein lipase, is essentially flexible, but has three defined secondary structural elements. The most significant one is an -helix between residues 67 and 74. The following three residues adopt a turn-like structure. Another turn of -helix is seen between residues 56 and 59. The effect of the solvent system on the secondary structure was studied by circular dichroism spectroscopy. The results show that the mixed aqueous 35% HFP solvent induces secondary structure of a very similar nature to the one induced by sodium dodecyl sulphate.Abbreviations Apo-CII Apolipoprotein CII - CD Circular Dichroism - DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine - DOPG 1,2-dioleoyl-sn-glycero-3-phosphoglycerol - HAc Acetic Acid - HFP 1,1,1,3,3,3-hexafluoro-2-propanol - ISPA Isolated Spin Pair Approximation - NMR Nuclear Magnetic Resonance - NOE Nuclear Overhauser Enhancement - NOESY Nuclear Overhauser Enhancement Spectroscopy - RMSD Root Mean Square Deviation - SDS Sodium Dodecyl Sulfate     

Deviation versus violation plots: A new technique for assessing the self-consistency of NMR data

Summary A new method has been developed to test the self-consistency of distance constraints derived from NOESY spectra. The technique is based on the premise that the further the atomic coordinates of any given structure vary from the correct structure, the more NOE violations will occur in that structure. This relationship is quantified by plotting the deviation of each structure against the sum of the residual NOE violations. This type of plot is called a DVplot, which is generated by the following means: first the experimental constraints are used to generate a set of structures, then the amount of deviation and violation is quantified for each structure. The deviation of each structure is derived from the root-mean-squared deviation (rmsd) between each structure and the average structure. Violations are quantified for each structure using the new terms S1, S2, and S3. These terms measure the sum of all residual NOE violations greater than 0.1 Å, 0.2 Å, and 0.3 Å, respectively. DVplots are used to show that for series of structures calculated from a single set of NMR constraints, there is an approximate linear correlation between the rmsd and each of the three sums, S1, S2, and S3. Furthermore, it is proposed that the x-intercepts derived from the three plots of S1, S2, and S3 will converge if the NOE constraints are selfconsistent. The new technique is applied to five different proteins using both experimental and simulated constraint sets.Abbreviations DVplots deviation versus violation plots - NOE a correlation between two protons observed by the nuclear Overhauser effect - r correlation coefficient - rmsd root-mean-squared deviation - S1, S2, and S3 measures of the sum of all NOE violations greater than 0.1 Å, 0.2 Å, and 0.3 Å, respectively     

Solution structure of a LewisX analogue by off-resonance 1H NMR spectroscopy without use of an internal distance reference

Summary A recent ¹H NMR method has been applied to the determination of the solution structure and internal dynamics of a synthetic mixed C/O trisaccharide related to sialyl Lewis^x. Varying the rf field offset in ROESY-type experiments enabled the measurement of longitudinal and transverse dipolar cross-relaxation rates with high accuracy. Assuming that for each proton pair the motion could be represented by a single exponential autocorrelation function, it was possible to derive geometrical parameters (r) and dynamic parameters _cp. With this assumption, 224 cross-relaxation rates have been transformed into 30 interproton distance constraints and 30 dipolar correlation times. The distance constraints have been used in a simulated-annealing procedure. This trisaccharide exhibits a structure close to the O-glycosidic analogue, but its flexibility seems highly reduced. On the basis of the determined structure and dynamics, it is shown that no conformational exchange occurs, the molecule existing in the form of a unique family in aqueous solution. In order to assess the quality of the resulting structures and to validate this new experimental procedure of distance extraction, we finally compare these solution structures to the ones obtained using three different sets of distances deduced from three choices of internal reference. It appears that this procedure allows the determination of the most precise and accurate solution.Abbreviations COSY correlation spectroscopy - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy; rmsd, root-mean-square deviation - ROESY rotating frame Overhauser enhancement spectroscopy - SLe^x sialyl Lewis^x - TOCSY total correlation spectroscopy     

The relative orientation of the fibronectin 6F11F2 module pair: A 15N NMR relaxation study

The structure of a pair of modules (⁶F1¹F2), that forms part of the collagen-binding region of fibronectin, is refined using heteronuclear relaxation data. A structure of the pair was previously derived from ¹H-¹H NOE and ³ J _HHN data [Bocquier et al. (1999) Structure, 7, 1451–1460] and a weak module–module interface, comprising Leu19 and Leu28, in ⁶F1, and Tyr68 in ²F1, was identified. In this study, the definition of the average relative orientation of the two modules is improved using the dependence of ¹⁵N relaxation on rotational diffusion anisotropy. This structure refinement is based on the selection of a subset of structures from sets calculated with NOE and ³ J _HHN data alone, using the quality of the fits to the relaxation data as the selection criterion. This simple approach is compared to a refinement strategy where ¹⁵N relaxation data are included in the force field as additional restraints [Tjandra et al. (1997) Nat. Struct. Biol., 4, 443–449].     

The activation mechanism of human porphobilinogen synthase by 2-mercaptoethanol: intrasubunit transfer of a reserve zinc ion and coordination with three cysteines in the active center

Human porphobilinogen synthase [EC.4.2.1.24] is a homo-octamer enzyme. In the active center of each subunit, four cysteines are titrated with 5,5-dithiobis(2-nitrobenzoic acid). Cys¹²², Cys¹²⁴ and Cys¹³² are placed near two catalytic sites, Lys¹⁹⁹ and Lys²⁵², and coordinate a zinc ion, referred to as a proximal zinc ion, and Cys²²³ is placed at the orifice of the catalytic cavity and coordinates a zinc ion, referred to as a distal zinc ion, with His¹³¹ . When the wild-type enzymes C122A (Cys¹²²Ala), C124A (Cys¹²⁴Ala), C132A (Cys¹³²Ala) and C223A (Cys²²³Ala) were oxidized by hydrogen peroxide, the levels of activity were decreased. Two cysteines were titrated with 5,5-dithiobis(2-nitrobenzoic acid) in the wild-type enzyme, while on the other hand, one cysteine was titrated in the mutant enzymes. When wild-type and mutant enzymes were reduced by 2-mercaptoethanol, the levels of activity were increased: four and three cysteines were titrated, respectively, suggesting that a disulfide bond was formed among Cys¹²², Cys¹²⁴ and Cys¹³² under oxidizing conditions. We analyzed the enzyme-bound zinc ion of these enzymes using inductively coupled plasma mass spectrometry with gel-filtration chromatography. The results for C223A showed that the number of proximal zinc ions correlated to the level of enzymatic activity. Furthermore, zinc-ion-free 2-mercaptoethanol increased the activity of the wild-type enzyme without a change in the total number of zinc ions, but C223A was not activated. These findings suggest that a distal zinc ion moved to the proximal binding site when a disulfide bond among Cys¹²², Cys¹²⁴ and Cys¹³² was reduced by reductants. Thus, in the catalytic functioning of the enzyme, the distal zinc ion does not directly contribute but serves rather as a reserve as the next proximal one that catalyzes the enzyme reaction. A redox change of the three cysteines in the active center accommodates the catch and release of the reserve distal zinc ion placed at the orifice of the catalytic cavity.     

De novo protein structure determination using sparse NMR data

We describe a method for generating moderate to high-resolution protein structures using limited NMR data combined with the ab initio protein structure prediction method Rosetta. Peptide fragments are selected from proteins of known structure based on sequence similarity and consistency with chemical shift and NOE data. Models are built from these fragments by minimizing an energy function that favors hydrophobic burial, strand pairing, and satisfaction of NOE constraints. Models generated using this procedure with 1 NOE constraint per residue are in some cases closer to the corresponding X-ray structures than the published NMR solution structures. The method requires only the sparse constraints available during initial stages of NMR structure determination, and thus holds promise for increasing the speed with which protein solution structures can be determined.     

Conformational analysis of α-d-Fuc-(1→4)-β-d-GlcNAc-OMe. One-dimensional transient NOE experiments and metropolis Monte Carlo simulations

Summary One-dimensional transient NOE build-up curves were measured for the synthetic disaccharide -d-Fuc-(14)--d-GlcNAc 1 utilizing Gaussian shaped pulses. Simulated build-up curves from Metropolis Monte Carlo simulations were compared to the experimental data. Disaccharide 1 is structurally related to methyl -d-maltoside in that it also contains an -(14) linkage, and it has the same configuration of groups around the glycosidic linkage. Analysis of NOEs in methyl -d-maltoside is restricted to those observed upon selective excitation of H1 because of severe spectral overlap. The situation is different in 1 where ¹H-NMR signals are well separated. Several interglycosidic NOEs were observed. The corresponding build-up curves allowed an accurate determination of the conformational preferences at the glycosidic linkage in 1. Comparison of experimental and theoretical NOE build-up curves showed clearly that rigid minimum-energy models cannot account for the experimental data. The best fit of experimental NOE build-up curves was obtained with theoretical curves from a 2×10⁶ step Metropolis Monte Carlo simulation with the temperature parameter set at 1000 K. Finally, it was observed that only the interglycosidic NOE H5/H6-pro-S significantly depends upon varying conformation distributions at the -(14)-glycosidic linkage, induced by choosing different temperature parameters for the Metropolis Monte Carlo simulations.     

Three-dimensional structure of α-conotoxin EI determined by 1H NMR spectroscopy H resonance assignments of α-conotoxin EI

-Conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom of Conus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ Torpedo, showing a novel selectivity profile when compared to other -conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between Thr⁹ and Met¹² that is stabilized by the Cys⁴-Cys¹⁰ disulfide bond and turns involving Cys⁴-Cys⁵ and Asn¹⁴-Pro¹⁵. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other 4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other 4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these 4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI.