Studies on solution NMR structure of brazzein——Secondary structure and molecular scaffold

Brazzein is a sweet-tasting protein isolated from the fruit of West African plantPentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far and is highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300 K) is presented. The complete sequence specific assignments of the individual backbone and sidechain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one alpha-helix (residues 21-29), one short 3(10)-helix (residues 14-17), two strands of antiparallel beta-sheet (residues 34-39, 44-50) and probably a third strand (residues 5-7) near the N-terminus. A comparative analysis found that brazzein shares a so-called 'cysteine-stabilized alpha-beta' (CSalphabeta) motif with scorpion neurotoxins, insect defensins and plant gamma - thionins. The significance of this multi-function motif, the possible active sites and the structural basis of themostability were discussed.     

Fucoidans and fucoidanases—focus on techniques for molecular structure elucidation and modification of marine polysaccharides

The research field of fucoidans (sulphated polysaccharides from algae) and fucoidanases was strongly developing in recent years. Several different fucoidans and a few fucoidan-degrading enzymes were isolated and characterised. A high potential is seen in the medical exploitation of the fucoidans and its degradation products. This review gives an overview about the research of the last 5 years concerning fucoidan characterisation and application as well as enzyme detection, characterisation and production.     

The NMR solution structure and characterization of pH dependent chemical shifts of the β-elicitin, cryptogein

The NMR structure of the 98 residue -elicitin, cryptogein, which induces a defence response in tobacco, was determined using 15N and 13C/15N labelled protein samples. In aqueous solution conditions in the millimolar range, the protein forms a discrete homodimer where the N-terminal helices of each monomer form an interface. The structure was calculated with 1047 intrasubunit and 40 intersubunit NOE derived distance constraints and 236 dihedral angle constraints for each subunit using the molecular dynamics program DYANA. The twenty best conformers were energy-minimized in OPAL to give a root-mean-square deviation to the mean structure of 0.82 Å for the backbone atoms and 1.03 Å for all heavy atoms. The monomeric structure is nearly identical to the recently derived X-ray crystal structure (backbone rmsd 0.86 Å for residues 2 to 97) and shows five helices, a two stranded antiparallel -sheet and an -loop. Using 1H,15N HSQC spectroscopy the pKa of the N- and C-termini, Tyr12, Asp21, Asp30, Asp72, and Tyr85 were determined and support the proposal of several stabilizing ionic interactions including a salt bridge between Asp21 and Lys62. The hydroxyl hydrogens of Tyr33 and Ser78 are clearly observed indicating that these residues are buried and hydrogen bonded. Two other tyrosines, Tyr47 and Tyr87, show pKa's >12, however, there is no indication that their hydroxyls are hydrogen bonded. Calculations of theoretical pKa's show general agreement with the experimentally determined values and are similar for both the crystal and solution structures.     

Studies on molecular evolution and structural features of double-headed inhibitors of α-amylase and trypsin in plants

Plant seeds usually have high concentrations of proteinase and amylase inhibitors. These inhibitors exhibit a wide range of specificity, stability and oligomeric structure. In this communication, we report analysis of sequences that show statistically significant similarity to the double-headed α-amylase/trypsin inhibitor of ragi (Eleusine coracana). Our aim is to understand their evolutionary and structural features. The 14 sequences of this family that are available in the SWISSPROT database form three evolutionarily distinct branches. The branches relate to enzyme specificities and also probably to the oligomeric state of the proteins and not to the botanical class of the plant from which the enzymes are derived. This suggests that the enzyme specificities of the inhibitors evolved before the divergence of commercially cultivated cereals. The inhibitor sequences have three regions that display periodicity in hydrophobicity. It is likely that this feature reflects extended secondary structure in these segments. One of the most variable regions of the polypeptide corresponds to a loop, which is most probably exposed in the native structure of the inhibitors and is responsible for the inhibitory property.     

Secondary structure of Qβ RNA

The complete set of possible secondary structures of a variant Qβ RNA sequenced by Schaffner has been found using a computer program which allows G-U pairing as well as the usual Watson-Crick A-U and G-C pairing. Of special interest are those secondary structures with the highest double-strandedness. Omitting G-U pairing, we find the structure with the maximum double-strandedness has a pairing of 62% and exhibits a similarity to the clover leaf structure of tRNA. Including G-U pairing, the complementary strands of RNA are asymmetrical. We find maximum pairings of 71% for both the plus and minus strands. These structures also exhibit a cloverleaf structure. A similar analysis has been carried out for the secondary structure of a larger Qβ variant sequenced by Mills, Kramer and Spiegelman, but in this case there are a large number of secondary structures with the same maximum number of pairs and it is therefore not possible to select a unique structure with the maximum double-strandedness.     

The crystal and molecular structure of α-laminaribiose octa-acetate

The crystal and molecular structure of octa-O-acetyl-α-laminaribiose is compared with those of disaccharides, and their acetylated derivatives, related to polysaccharides of the laminarin and curdlan type. Marked differences are found in the endo- and exo-cyclic torsion angles as well as in the molecular geometry of the glycosidic linkage. The conformation of AcO-6′ provides the first experimental evidence of the theoretically predicted gauche-trans-gauche⁻ conformation. This conformational change, together with the α-orientation of AcO-1, alters the overall shape of the molecule and influences the packing features. The effect of the acetylation is also examined in terms of calculated conformational energy maps.     

The other kind of biological NMR—Studies of enzyme-substrate interactions

NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in drug metabolism. Cytochromes P₄₅₀ play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate complexes. The other two enzymes, chloramphenicol acetyltransferase and β-lactamase, are responsible for bacterial resistance to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme A bound to the enzyme, while in the case of β-lactamase the pK of a specific lysine residue at the active site has been determined, providing valuable information on the catalytic mechanism. Special issue dedicated to Dr. Herman Bachelard.     

1H, 13C and 15N NMR assignments and solution secondary structure of rat Apo-S100β

Summary The ¹H, ¹³C and ¹⁵N NMR assignments of the backbone and side-chain resonances of rat S100 were made at pH 6.5 and 37°C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE correlations, together with amide exchange rate and ¹H, ¹³C and ¹³C chemical shift data, provided extensive secondary structural information. Thus, the secondary structure of S100 was determined to comprise four helices (Leu³-Ser¹⁸, helix I; Lys²⁹-Leu⁴⁰, helix II; Gln⁵⁰-Glu⁶², helix III; and Phe⁷⁰-Ala⁸³, helix IV), four loops (Gly¹⁹-His²⁵, loop I; Ser⁴¹-Glu⁴⁹, loop II; Asp⁶³-Gly⁶⁶, loop III; and Cys⁸⁴-Glu⁹¹, loop IV) and two -strands (Lys²⁶-Lys²⁸, -strand I and Glu⁶⁷-Asp⁶⁹, -strand II). The -strands were found to align in an antiparallel manner to form a very small -sheet. This secondary structure is consistent with predictions that S100 contains two helix-loop-helix Ca²⁺-binding motifs known as EF-hands. The alignment of the -sheet, which brings the two EF-hand domains of S100 into close proximity, is similar to that of several other Ca²⁺ ion-binding proteins.     

Effect of Zn ion on the structure and electronic states of Aβ nonamer: molecular dynamics and ab initio molecular orbital calculations

Aggregates of amyloid-beta proteins (Aβ) have been recognised to be intimately related to pathogenesis of Alzheimer’s disease (AD). Indeed, Aβ aggregates of various sizes from dimers to fibrils were found in the brains of AD patients, and these aggregates can be self-organised. Since abnormal accumulation of metal ions such as Zn, Cu and Fe was also observed in the brains, the association between Aβ aggregations and these metal ions has been studied widely. In the present study, to elucidate the influence of Zn ions on the stability of Aβ aggregates, we performed molecular dynamics (MD) simulations and ab initio fragment molecular orbital (FMO) calculations on the Aβ nonamers with and without Zn ions and investigated the change in its structure and electronic states induced by Zn ions at atomic and electronic levels. The MD simulations revealed that Aβ nonamer cannot keep its symmetry structure, whereas Aβ nonamer with Zn ions keeps the structure. The FMO results indicated that electrostatic interactions among the charged amino-acid residues of Aβ nonamer are significantly changed by the influence of Zn ions to stabilise Aβ nonamer. These results provide useful information for proposing novel compounds, which binds specifically to Aβ and inhibits the Aβ aggregation.     

Studies on chemical structure modification and structureactivity relationship of derivatives of gambogic Acid at c(39)

The natural product gambogic acid exhibits high potency in inhibiting cancer cell lines. Rational medicinal modifications on gambogic acid will improve its physicochemical properties and drug-like characters. To investigate the structure?activity relationship of gambogic acid and also to find rational modification position on its chemical skeleton, we designed, synthesized, and characterized 16 derivatives of gambogic acid that were modified at C(39). The structure?activity relationships (SARs) were discussed. The anti-proliferation data were accquired through MTT (=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assays of A549, BGC823, U251, HepG2, and MDA-MB-231 cancer cell lines. Most of the synthesized compounds showed strong inhibitory effects. The SAR study revealed that derivatives with aliphatic amino moieties at C(39) were more potent than those with other substituents. The C(39) position can undergo different kinds of chemical modifications without leading to loss of activity. Compounds 4 and 6 can serve as potential lead compounds for further development of new anticancer drugs.     

Fibre and molecular structure of thymidylyl-3′,5′-deoxyadenosine

X-ray diffraction and molecular model building studies of an ordered structure of thymidylyl-3′,5′-deoxyadenosine which gives fibre-type diffraction patterns, are consistent with a seven-residues per turn, left-handed structure in which the adenine of one molecule and the thymine of the next are linked together by Hoogsteen type of hydrogen bonds. The structure thus resembles a macromolecule in which units are linked together by hydrogen bonds and stabilized by base stocking. Both nucleosides in the basic molecule are in the anti conformation and both sugar rings have C3′-endo puckers. The C5′-05′ bond of the deoxyadenosine is trans relative to C4′-C3′ and the conformations about the P-03′ and P-05′ bond are gauche^?, trans.     

How is an NMR structure best defined? An analysis of molecular dynamics distance-based approaches

Summary Model studies on the macrocyclic immunosuppressive agent FK506 challenge traditional approaches to defining a structure from data collected during a 2D NMR experiment. A variety of joint molecular dynamics/NMR-distance refinement methodologies are characterized. From the results it is clear that the traditional presentation of an NMR structure as a single representative minimized conformation or as a fairly tight envelope of conformers best meeting the imposed restraints can be misleading; a greater emphasis is required on dynamics and on the fact that an NMR structure represents a time average.     

14. Studies on Poliomyelitis Virus—Concentration and Purification of the Virus

ABSTRACT

For the purpose of determining the immunogenic potency of polio virus, relatively large amounts of concentrated virus material were prepared which had titres of the order of 10¹⁰ T.C.I.D.jo per ml. These were obtained by pervaporating large quantities of tissue culture fluid containing approximately 10⁶⁵ T.C.I.D.JQ per ml.     

Energetics and structure of alanine-rich α-helices via adaptive steered molecular dynamics

The energetics and hydrogen bonding profiles of the helix-to-coil transition were found to be an additive property and to increase linearly with chain length, respectively, in alanine-rich α-helical peptides. A model system of polyalanine repeats was used to establish this hypothesis for the energetic trends and hydrogen bonding profiles. Numerical measurements of a synthesized polypeptide Ac-Y(AEAAKA)_kF-NH₂ and a natural α-helical peptide a2N (1–17) provide evidence of the hypothesis’s generality. Adaptive steered molecular dynamics was employed to investigate the mechanical unfolding of all of these alanine-rich polypeptides. We found that the helix-to-coil transition is primarily dependent on the breaking of the intramolecular backbone hydrogen bonds and independent of specific side-chain interactions and chain length. The mechanical unfolding of the α-helical peptides results in a turnover mechanism in which a 3₁₀-helical structure forms during the unfolding, remaining at a near constant population and thereby maintaining additivity in the free energy. The intermediate partially unfolded structures exhibited polyproline II helical structure as previously seen by others. In summary, we found that the average force required to pull alanine-rich α-helical peptides in between the endpoints—namely the native structure and free coil—is nearly independent of the length or the specific primary structure.     

Evolutionary modifications of molecular structure of ATP-synthase γ-subunit

The ATP-synthase γ-subunit (FoF1) belongs to the rotor part of this oligomeric complex. Catalytic hydrolysis of adenosine triphosphate (ATP) is accompanied by rotation of γ-polypeptide inside the sphere formed by six subunits (αβ)₃ of the enzyme. The γ-subunit regulates ATPase and ATP-synthase activities of the FoF1. In the present work, evolutionary and reverse changes of this regulatory polypeptide and their effect on properties of the enzyme are studied. It is suggested that elongation of the γ-subunit globular part had resulted from the atpC intragene duplication in the process of adaptive evolution. The evolved fragment participates in light regulation of the chloroplast ATP-synthase.     

5.5 Å crystallographic structure of penicillin β-lactamase and radius of gyration in solution

An electron density map of crystalline R-TEM Escherichia coli β-lactamase (penicillinase) has been calculated from X-ray diffraction data at 5.5 Å resolution with protein phases based on Friedel mates from a high-quality samarium derivative. The mean figure of merit for 854 independent reflections is 0.75. The monomeric molecule is slightly ellipsoidal and contains one and possibly two regions of α-helix which are 25 Å long. The Crystallographic search for the substrate binding site has so far been inconclusive. The radius of gyration of the enzyme in solution at pH 7 is 17.1 ± 1.0 Å from small-angle X-ray scattering measurements. This compares with 18.6 å calculated from the low-resolution electron density map of the molecule in the crystal.