Structure and conformational heterogeneity of the weak toxin from the cobra Naja kaouthia venom

Resonances in the two-dimensional 1H NMR spectra of a weak toxin (WTX) from the venom of cobra Naja kaouthia for all 65 amino acid residues were assigned. The amino acid sequence of WTX, determined by the sequentional assignment of spin systems, was found to be similar to that of the CM-9a toxin from the N. kaouthia venom. Unlike CM-9a, WTX contains an additional Trp36 residue; Lys50 and Tyr52 are interchanged; and there is a Thr residue in place of Arg2. For some residues of WTX, the presence of two components of approximately equal intensities in the spectra was shown, which is explained by the conformational heterogeneity of the polypeptide owing to the cis-trans isomerization of the peptide bond Arg32-Pro33. The data (contacts of the nuclear Overhauser effect, constants of spin-spin coupling of protons, and rates of exchange of amide protons by deuterium of the solvent) made it possible to determine the secondary structure of two forms of WTX, which is characterized by the presence of two antiparallel beta-sheets, one of which consists of two strands (regions 1-5 and 13-17) and the other, of three strands (regions 23-28, 38-43, and 55-59).     

New weak toxins from the cobra venom

A protein with M 7485 Da containing five disulfide bonds was isolated from the venom of cobra Naja oxiana using various types of liquid chromatography. The complete amino acid sequence of the protein was determined by protein chemistry methods, which permitted us to assign it to the group of weak toxins. This is the first weak toxin isolated from the venom of N. oxiana. In a similar way, two new toxins with M 7628 and 7559 Da, which fall into the range of weak toxin masses, were isolated from the venom of the cobra N. kaouthia. The characterization of these proteins using Edman degradation and MALDI mass spectrometry has shown that one of these proteins is a novel weak toxin, and the other is the known weak toxin WTX with an oxidized methionine residue in position 9. Such a modification was detected in weak toxins for the first time. A study of the biological activity of the toxin from N. oxiana showed that, like other weak toxins, it can be bound by α7 and muscle-type nicotinic acetylcholine receptors.     

Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional “three-finger” snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by “three-finger” snake neurotoxins.     

Toxin III of the scorpionAndroctonus australis Hector: Proton nuclear magnetic resonance assignments and secondary structure

Summary ¹H NMR has been applied to a3.5 mM, pH 5.4, solution of toxin III (64 amino acids) from venom of the scorpionAndroctonus australis Hector. The resonance assignment strategy began by applying a generalized main-chain directed method for rapid identification and resonance assignments of secondary structures. The remaining resonances were assigned by the sequential method. Major structural features include a helix of 2 1/2 turns (residues 20–28) which is linked by two disulfide bridges to the central strand of a triple-stranded antiparallel -sheet. Turns were identified at residues 15–17, 47–49 and also at residues 51–53. Numerous NOEs have been observed between hydrophobic residues which suggest the presence of a hydrophobic core; these include Leu³⁷, Leu²³, Val⁴⁷, Tyr¹⁴, Trp⁴⁵ and Tyr⁵. The Trp⁴⁵ and Tyr⁵ rings lie orthogonal to one another. No crystal structure has been solved for this AaH III toxin. Comparisons are made with other members of the scorpion toxin family.Thenomenclature used is similar to that described by Wütrich, 1986.     

Assignments of1H−15N magnetic resonances and identification of secondary structure elements of the λ-cro repressor

Summary The assignments of¹H–¹⁵N magnetic resonances of the -cro repressor are presented. Individual¹⁵N-amino acids were incorporated into the protein, or it was uniformly labeled with¹⁵N. For the¹³C–¹⁵N double-labeling experiments,¹³C-amino acids were incorporated into the uniformly¹⁵N-labeled protein. All the amide¹H–¹⁵N resonances could be assigned with such specific labeling, and sequential connectivities obtained by two-dimensional (2D)¹H–¹⁵N reverse correlation spectroscopies and three-dimensional (3D)¹H/¹⁵N NOESY-HMQC spectroscopy. Conventional 2D¹H–¹H correlation spectroscopies were applied to the assignment of the side-chain protons. Some of the¹H resonance assignments are inconsistent with those previously reported [Weber, P.L., Wemmer, D.E. and Reid, B.R. (1985)Biochemistry,24, 4553–4562]. The sequential NOE connectivities and H-D exchange rates indicate several elements of the secondary structure, including -helices consisting of residues 8–15, 19–25 and 28–37, and three extended strands consisting of residues 4–7, 39–45 and 49–55. Based on several long-range NOEs, the three extended strands could be combined to form an antiparallel -sheet. The amide proton resonances of the C-terminal residues except Ala⁶⁶ (residues 60–65) were hardly observed at neutral pH, indicating that the arm is flexible. The identified secondary structure elements in solution show good agreement with those in the crystal structure of the cro protein [Anderson, W.F., Ohlendorf, D.H., Takeda, Y. and Matthews, B.W. (1981)Nature,290, 754–758].     

Covalent Structure of Botulinum Neurotoxin Type E: Location of Sulfhydryl Groups, and Disulfide Bridges and Identification of C-Termini of Light and Heavy Chains

Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an 150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992), Biochem. Biophys. Res. Commun. 183, 107–113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein—at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)—is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419–421 (Gly–Ile–Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1–Lys 418 constitute the 50–kDa light chain, Lys 422–Lys 1251 constitute the 100–kDa heavy chain; Cys 411–Cys 425 and Cys 1196–Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The 150–kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without ^I4C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nucleotide sequence.     

Resonant recognition model of neuropeptide Y family: hot spot amino acid distribution in the sequences

The resonant recognition model is used to predict structurally and functionally important amino acid residues (so-called hot spots) in the neuropeptide Y (NPY) family. Thirty-three polypeptides belong to this family. All of them consist of 36 amino acids. The model predicts that residues 10 and 28 in the polypeptides are hot spots. In the 33 polypeptides, most of the amino acids at residue 10 are acidic amino acids, glutamic acid and aspartic acid. Other minor amino acids, serine, glycine, and proline, have high probabilities of -turn occurrence. Amino acids at residue 28 are all branched hydrophobic amino acids, isoleucine, leucine, and valine. The profile for predicting hot spots indicates repeating patterns of residues 1–18 and residues 19–36. Absolute values at residue i and residue i + 18 are the same, but these residues have opposite signs. Therefore the model of the NPY family predicts hot spots concerning a combination of residue i and residue i + 18.     

Proton uptake upon quinone reduction in bacterial reaction centers: IR signature and possible participation of a highly polarizable hydrogen bond network

The light-induced Q _A ^– /Q_A FTIR difference spectra of Rb. sphaeroides and Rp. viridis show very broad positive bands of small amplitude peaking around 2750 cm^–1. Upon ¹H/²H exchange these bands shift to about 2150 cm^–1. Similarly, the Q _B ^– /Q_B spectra exhibit broad continuum bands at 2600 and 2800 cm^–1 shifting to 2100 and 2200 cm^–1 in ²H₂O for Rb. sphaeroides and Rp. viridis, respectively. These continuum bands are tentatively interpreted in terms of highly polarizable hydrogen bonds in a large web of polar bonds involving cofactors, amino acid residues, and structured water molecules. As a working hypothesis, we propose that the protons participating in this web redistribute upon quinone reduction, increasing their concentration around the newly formed charged species, and leading to net proton uptake. Assuming that the precise localization of the mobile protons is dependent on the local electrostatic, this model can explain the apparent discrepancies between some results of FTIR experiments and of electrostatic calculations. Notably, it could help rationalize the observation that mobile protons tend to localize on Glu L212 upon Q_B reduction in Rb. sphaeroides, while for Q_B reduction in Rp. viridis and for Q_A reduction in both Rb. sphaeroides and Rp. viridis, proton uptake by a small number of carboxylic residues is not supported by the FTIR data.     

Analysis of mobility of protein side chains by spin label technique

Five spin labeled derivatives of a neurotoxin from cobra venom were analyzed by the earlier suggested method. The procedure was adjusted to the complex motional behaviour of the label. Each protein derivative carried covalently bound spin label on different lysine residues. In two derivatives, at positions Lys44 and Lys46, the labels were strongly mobile, whereas for other three derivatives modified at Lys15, Lys25 and Lys26 the label was less mobile with respect to the protein molecule, which made possible determination of the rotational correlation time of the protein molecule (2.8±0.3 ns). The rotational correlation time was in good agreement with the calculated value for the rigid sphere of the corresponding molecular weight. On the basis of the estimate of the anisotropic motion degree, it was found from the order parameter S that the label mobility increases in the following series of lysine residues: Lys26, Lys25, Lys15, Lys46, and Lys44. From the analysis of positions of outer wide peaks in ESR spectra obtained by varying temperature and viscosity of the medium, we determined the parameters for computer simulation. The theoretical and experimental spectra were found to be in good agreement.Nomenclature rotational correlation time of the protein molecule - _l rotational correlation time of the spin label - 2A _Z , 2A the rigid limit distance between OWP and IWP, respectively, for ESR spectra of spin labeled proteins - 2, 2 the averaged limit distance between the OWP and IWP correspondingly mobile spin label to respect of protein moiety with; = - 2A',2A distance between OWP and IWP in the ESR spectra of spin labeled proteins for any T and media Abbreviations SL spin label - NT neurotoxin II from cobra venom - NT-SL-Lys44 neurotoxin spin labeled at Lys44 residue - OWP outer wide peaks in the immobilized ESR spectra - IWP inner wide peaks in the immobilized ESR spectra - WL the residual linewidth     

Heteronuclear three-dimensional NMR spectroscopy of a partially denatured protein: The A-state of human ubiquitin

Summary Human ubiquitin is a 76-residue protein that serves as a protein degradation signal when conjugated to another protein. Ubiquitin has been shown to exist in at least three states: native (N-state), unfolded (U-state), and, when dissolved in 60% methanol:40% water at pH 2.0, partially folded (A-state). If the A-state represents an intermediate in the folding pathway of ubiquitin, comparison of the known structure of the N-state with that of the A-state may lead to an understanding of the folding pathway. Insights into the structural basis for ubiquitin's role in protein degradation may also be obtained. To this end we determined the secondary structure of the A-state using heteronuclear three-dimensional NMR spectroscopy of uniformly ¹⁵N-enriched ubiquitin. Sequence-specific ¹H and ¹⁵N resonance assignments were made for more than 90% of the residues in the A-state. The assignments were made by concerted analysis of three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets. Because of ¹H chemical shift degeneracies, the increased resolution provided by the ¹⁵N dimension was critical. Analysis of short- and long-range NOEs indicated that only the first two strands of -sheet, comprising residues 2–17, remain in the A-state, compared to five strands in the N-state. NOEs indicative of an -helix, comprising residues 25–33, were also identified. These residues were also helical in the N-state. In the N-state, residues in this helix were in contact with residues from the first two strands of -sheet. It is likely, therefore, that residues 1–33 comprise a folded domain in the A-state of ubiquitin. On the basis of ¹H chemical shifts and weak short-range NOEs, residues 34–76 do not adopt a rigid secondary structure but favor a helical conformation. This observation may be related to the helix-inducing effects of the methanol present. The secondary structure presented here differs from and is more thorough than that determined previously by two-dimensional ¹H methods [Harding et al. (1991) Biochemistry, 30, 3120–3128].     

Motional properties of a pentasaccharide containing a 2,6-branched mannose residue as studied by 13C nuclear spin relaxation

Summary ¹³C relaxation data obtained at three different magnetic fields, 9.4, 11.8 and 14.1 T, and at two temperatures, 303 and 318 K, are reported for the pentasaccharide p-trifluoroacetamidophenyl 2,6-di-O-[-d-galactopyranosyl-(14)-O-2-acetamido-2-deoxy--d-glucopyranosyl]-d-mannopyranoside. The pentasaccharide consists of two disaccharide units, attached at position 2 and 6 to the central mannopyranoside residue. The relaxation data were interpreted with the Lipari-Szabo model-free approach. For the central mannose residue in the molecule a high order parameter (S²=0.91) was found and the relaxation data could be interpreted with the truncated form of the Lipari-Szabo model. The motional behavior of the two 2-acetamido-2-deoxy-glucopyranoside residues was found to differ. The one attached at the primary hydroxylic position displayed more extensive local motion (S²=0.75–0.77) than the one attached at the secondary hydroxylic position (S²=0.83–0.85). More extensive local motion for the two outer galactopyranoside residues was found (S²=0.56–0.59), but no significant difference in motional behavior between the two residues could be observed. Analysis of the relaxation data for the exocyclic carbons confirmed the results for the rings. For the mannose C6, the same motional parameters as obtained for the substituting 2-acetamido-2-deoxy-glucopyranoside residue were found. The two exocyclic carbons on the 2-acetamido-2-deoxy-glucopyranoside residues showed more extensive local motion, with lower order parameters (S²=0.59–0.66).To whom correspondence should be addressed.     

Graspases--a special group of serine proteases of the chymotrypsin family that has lost a conserved active site disulfide bond

In this report we propose a new approach to classification of serine proteases of the chymotrypsin family. Comparative structure–function analysis has revealed two main groups of proteases: a group of trypsin-like enzymes and graspases (granule-associated proteases). The most important structural peculiarity of graspases is the absence of conservative active site disulfide bond Cys191–Cys220. The residue at position 226 in the S1-subsite of graspases is responsible for substrate specificity, whereas the residue crucial for specificity in classical serine proteases is located at position 189. We distinguish three types of graspases on the base of their substrate specificity: 1) chymozymes prefer uncharged substrates and contain an uncharged residue at position 226; 2) duozymes possess dual trypsin-like and chymotrypsin-like specificity and contain Asp or Glu at 226; 3) aspartases hydrolyze Asp-containing substrates and contain Arg residue at 226. The correctness of the proposed classification was confirmed by phylogenic analysis.     

Characteristic moisture curves and maximum water content of two crop residues

Limited data are available relating water potential () to crop residue water content (), although this relationship is important to study decomposition and moisture retention of the residue layer in no-till systems and other agricultural situations where residues are used. The objectives of this study were (i) to determine the characteristic moisture curves of rye (Secale cereale L.) and clover residues (Trifolium incarnatum L.), and (ii) to determine residue characteristics that can predict maximum water content of crop residues. Air-dried residues were separated into leaves and stems, cut into 0.5 cm length pieces and saturated with distilled water. Pieces of the drained residues were dried to various water contents in the laboratory and then transferred into thermocouple psychrometer chambers. Characteristic moisture functions of the type = a ^–b, where a and b are empirical constants, were fitted to the data. The characteristic moisture curves had a similar shape to that of a Cecil sandy loam soil used as an example; however, while plant residues were able to retain up to 4.3 g H₂O g^–1, the mineral soil retained only 0.22 g H₂O g^–1. Soluble carbohydrate concentration can be used as a practical index to estimate maximum water content of residues, given the good relationship between both variables (R ² = 0.92).     

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

Summary The assignments of the ¹H, ¹⁵N, ¹³CO and ¹³C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled ¹⁵N- and ¹⁵N-/¹³C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic ¹H and ¹³C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as ³J_H n _H coupling constants, amide exchange and ¹³C and ¹³C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.     

New Substrates for Enteropeptidase: I. Biologically Active Hepta-, Octa-, and Nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue if less than four negatively charged amino acid residues are in positions P ₂–P ₅ of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR VYIHPF) and the Hb(2–8) (LTAEEK A) and Hb(1–9) (MLTAEEK AA) peptides of the cattle hemoglobin -chain. The K _m values for all the substrates (10^–3 M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the –DDDDK– full-size linker (K _m 10^–4 M). The k _cat values for AT and Hb(2–8) were also close and low (30 min^–1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2–8) peptide by one amino acid residue from both its N- and C-termini results in a dramatic increase in the catalytic efficiency of the hydrolysis: the k _cat value for Hb(1–9) is 1510 min^–1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

New paradoxical three-finger toxin from the cobra <Emphasis Type="Italic">Naja kaouthia</Emphasis> venom: Isolation and characterization

A new three-finger toxin nakoroxin was isolated from the cobra Naja kaouthia venom, and its complete amino acid sequence was established. Nakoroxin belongs to the group of “orphan” toxins, data on the biological activity of which are practically absent. Nakoroxin shows no cytotoxicity and does not inhibit the binding of α-bungarotoxin to nicotinic acetylcholine receptors of muscle and α7 types. However, it potentiates the binding of α-bungarotoxin to the acetylcholine-binding protein from Lymnaea stagnalis. This is the first toxin with such an unusual property.     

An orientation-selected ENDOR and HYSCORE study of the Ni-C active state of Desulfovibrio vulgaris Miyazaki F hydrogenase

Electron nuclear double resonance (ENDOR) and hyperfine sublevel correlation spectroscopy (HYSCORE) are applied to study the active site of catalytic [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F in the reduced Ni-C state. These techniques offer a powerful tool for detecting nearby magnetic nuclei, including a metal-bound substrate hydrogen, and for mapping the spin density distribution of the unpaired electron at the active site. The observed hyperfine couplings are assigned via comparison with structural data from X-ray crystallography and knowledge of the complete g-tensor in the Ni-C state (Foerster et al. (2003) J Am Chem Soc 125:83–93). This is found to be in good agreement with density functional theory calculations. The two most strongly coupled protons (a_iso=13.7, 11.8 MHz) are assigned to the -CH₂ protons of the nickel-coordinating cysteine 549, and a third proton (a_iso=8.9 MHz) is assigned to a -CH₂ proton of cysteine 546. Using D₂O exchange experiments, the presence of a hydride in the bridging position between the nickel and iron—recently been detected for a regulatory hydrogenase (Brecht et al. (2003) J Am Chem Soc 125:13075–13083)—is experimentally confirmed for the first time for catalytic hydrogenases. The hydride exhibits a small isotropic hyperfine coupling constant (a_iso=–3.5 MHz) since it is bound to Ni in a direction perpendicular to the z-axis of the Ni orbital. Nitrogen signals that belong to the nitrogen N of His-88 have been identified. This residue forms a hydrogen bond with the spin-carrying Ni-coordinated sulfur of Cys-549. Comparison with other hydrogenases reveals that the active site is essentially the same in all proteins, including a regulatory hydrogenase.     

Specific assignment of resonances in the 1H-NMR spectrum of the polypeptide toxin I from Anemonia sulcata

The assignment of a large number of resonances in the 300-MHz ¹H-nmr spectrum of the polypeptide neurotoxin Anemonia sulcata toxin I is described. The initial identification of spin systems is made using both one- and two-dimensional nmr spectra. The subsequent assignment of these spin systems to specific residues in the molecule is based largely on the observation in two-dimensional spectra of through-space connectivities between H^α and NH resonances from adjacent residues in the amino acid sequence. Using these techniques, the full spin systems of 22 residues are specifically assigned, together with partial assignments for a further 8. Many of the spin systems from the remaining 16 residues have been defined, although not yet specifically assigned. From the pattern of through-space connectivities between protons from adjacent residues in the sequence, some inferences may be drawn concerning the secondary structure of this polypeptide in aqueous solution.