Molecular conformation of alamethicin in dimethylsulfoxide solution A two-dimensional n.m.r. study

The solution conformation of alamethicin, a 20-residue antibiotic peptide, has been investigated using two-dimensional n.m.r. spectroscopy. Complete proton resonance assignments of this peptide have been carried out using COSY, SUPERCOSY, RELAY COSY and NOESY two-dimensional spectroscopies. Observation of a large number of nuclear Overhauser effects between sequential backbone amide protons, between backbone amide protons and CβH protons of preceding residues and extensive intramolecular hydrogen bonding patterns of NH protons has established that this polypeptide is in a largely helical conformation. This result is in conformity with earlier reported solid state X-ray results and a recent n.m.r. study in methanol solution (Esposito et al. (1987) Biochemistry26, 1043-1050) but is at variance with an earlier study which favored an extended conformation for the C-terminal half of alamethicin (Bannerjee et al.

Conformational analysis of the right-hand twisted antiparallel β-Structure

The conformational analysis of a pair of two-linked peptide units in the anti-parallel arrangement is reported here with a view to study the effect of association of one chain with the other. The pair of two-linked peptide units were fixed in space through the hydrogen bonds between them, in accordance with certain hydrogen bond criteria. Model building was undertaken to ascertain whether the proximity of the side-chains could be used to eliminate any one of the right-hand twisted, left-hand twisted or regular β-structures. Stereochemically, it was found possible with all of them. The preference for a right-hand twisted β-structure, however, was indicated by the classical energy calculations. The relevance of the results thus obtained is discussed in the context of the preferential right-hand twist of the β-pleated sheets present in globular proteins. The agreement between the minimum energy conformations obtained for the pair of two-linked peptide units and the globular protein data is also indicated.

Successive refinement of structures with data of increasing resolution: a theoretical study for triclinic space groups

The method of least squares could be used to refine an imperfectly related trial structure by adoption of one of the following two procedures: (i) using all the observed at one time or (ii) successive refinement in stages with data of increasing resolution. While the former procedure is successful in the case of trial structures which are sufficiently accurate, only the latter has been found to be successful when the mean positional error (i.e.<|[Delta]r|>) for the atoms in the trial structure is large. This paper makes a theoretical study of the variation of the R index, mean phase-angle error, etc. as a function of <|[Delta]r|> for data corresponding to different esolutions in order to find the best refinement procedure [i.e. (i) or (ii)] which could be successfully employed for refining trial structures in which <|[Delta]r|> has large, medium and low values. It is found that a trial structure for which the mean positional error is large could be refined only by the method of successive refinement with data of increasing resolution.

Some implications of an alternative structure for DNA

We have constructed a space-filling (Corey-Pauling-Koltun) model of an alternative structure for DNA. This structure is not a double helix, but consists of a pair of polynucleotide strands lying side by side and held together by Watson-Crick base pairing. Each of the two strands has alternating right- and left-handed helical segments approximately five base pairs in length. Sugar residues in alternating segments along a strand point in opposite directions. A structure slightly different from the present one proposed earlier by ourselves and another group and in which sugars in a strand all point in the same direction is ruled out. The present structure yields natural solutions to the problems of supercoiling of DNA and of strand separation during DNA replication. This model is energetically more favorable than the double helix.

Vibrational analysis of boldine hydrochloride using QM/MM approach

A complete vibrational analysis was performed on the molecular structure of boldine hydrochloride using QM/MM method. The equilibrium geometry, harmonic vibrational frequencies and infrared intensities were calculated by QM/MM method with B3LYP/6-31G(d) and universal force field (UFF) combination using ONIOM code. We found the geometry obtained by the QM/MM method to be very accurate, and we can use this rapid method in place of time consuming ab initio methods for large molecules. A detailed interpretation of the infrared spectra of boldine hydrochloride is reported. The scaled theoretical wave numbers are in perfect agreement with the experimental values. The FT-IR spectra of boldine hydrochloride in the region 4000-500 cm(-1) were recorded in CsI (solid phase) and in chloroform with concentration 5 and 10 mg/ml.

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (ICFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K-D similar to 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access In recent years, IGFBPs have been implicated in a variety of cancers However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Eschericha coli Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E coli and first structural characterization of a full-length IGFBP (C) 2010 Elsevier Inc. All rights reserved

Crystallization and preliminary X-ray studies of a galactose-specific lectin from the seeds of bitter gourd (Momordica charantia)

A galactose-specific lectin from the seeds of bitter gourd (Momordica charantia) is a four-chain type II ribosome-inactivating protein (RIP) resulting from covalent association through a disulfide bridge between two identical copies of a two-chain unit. The available structural information on such four-chain RIPs is meagre. The bitter gourd lectin was therefore crystallized for structural investigation and the crystals have been characterized. It is anticipated that the structure of the orthorhombic crystals will be analysed using molecular replacement by taking advantage of its sequence, and presumably structural, homology to normal two-chain type II RIPs.

Crystallization and preliminary X-ray studies of the C-terminal domain of Mycobacterium tuberculosis LexA

The C-terminal domain of Mycobacterium tuberculosis LexA has been crystallized in two different forms. The form 1 and form 2 crystals belonged to space groups P3(1)21 and P3(1), respectively. Form 1 contains one domain in the asymmetric unit, while form 2 contains six crystallographically independent domains. The structures have been solved by molecular replacement.

Nerve growth factor induced turnover of phosphatidylinositol in rat superior cervical ganglia

The addition of nerve growth factor to organ cultures of superior cervical ganglia from immature rats specifically stimulated the incorporation of 32P-orthophosphate into phosphatidylinositol fraction. Equimolar concentrations of other hormones such as insulin, glucagon, thyroxine and growth hormone did not cause any stimulation of the incorporation of 14C-myoinositol into phosphatidylinositol. The stimulation of phosphatidylinositol turnover was observed over a concentration of nerve growth factor ranging from 10?10M to 10?7M. Nerve growth factor specific �inositide effect� was found to be sensitive to nerve growth factor antibody, 2,4-dinitrophenol, a high concentration of bovine growth hormones but not to Actinomycin D. The physiological significance of this finding in relation to nerve growth factor action in this target tissue is discussed.NGF, Nerve Growth Factor; SCG, Superior Cervical Ganglia; PI, Phosphatidylinositol

Purification and characterization of a beta-glucosidase and endocellulase from Humicola insolens

A .beta.-glucosidase and an endocellulase were purified from the culture filtrates of a thermophilic cellulolytic fungus Humicola insolens. Both the preparations were homogeneous by PAGE, ultracentrifugation and gel filtration (Mr 45,000). Ouchterlony immunodiffusion showed complete cross reactivity between the antibodies and the two enzyme antigens, indicating the presence of a common epitope on the two enzyme proteins. The two enzymes, however, differ in their amino acid composition and their substrate specificity. .beta.-Glucosidase acts on p-nitrophenyl .beta.-D-glucopyranoside and hydrolyses cellulose to release mainly glucose and small amounts of cellobiose from the non-reducing end. On the other hand, endocellulase hydrolyses cellulose to release cellopentaose, cellotetraose, cellotriose along with cellobiose and glucose and also hydrolyses larch wood xylan.