Identification of helix capping and β-turn motifs from NMR chemical shifts

We present an empirical method for identification of distinct structural motifs in proteins on the basis of experimentally determined backbone and ¹³C^β chemical shifts. Elements identified include the N-terminal and C-terminal helix capping motifs and five types of β-turns: I, II, I′, II′ and VIII. Using a database of proteins of known structure, the NMR chemical shifts, together with the PDB-extracted amino acid preference of the helix capping and β-turn motifs are used as input data for training an artificial neural network algorithm, which outputs the statistical probability of finding each motif at any given position in the protein. The trained neural networks, contained in the MICS (motif identification from chemical shifts) program, also provide a confidence level for each of their predictions, and values ranging from ca 0.7–0.9 for the Matthews correlation coefficient of its predictions far exceed those attainable by sequence analysis. MICS is anticipated to be useful both in the conventional NMR structure determination process and for enhancing on-going efforts to determine protein structures solely on the basis of chemical shift information, where it can aid in identifying protein database fragments suitable for use in building such structures.     

Type I and II β-turns prediction using NMR chemical shifts

A method for predicting type I and II β-turns using nuclear magnetic resonance (NMR) chemical shifts is proposed. Isolated β-turn chemical-shift data were collected from 1,798 protein chains. One-dimensional statistical analyses on chemical-shift data of three classes β-turn (type I, II, and VIII) showed different distributions at four positions, (i) to (i + 3). Considering the central two residues of type I β-turns, the mean values of C_ο, C_α, H_N, and N_H chemical shifts were generally (i + 1) > (i + 2). The mean values of C_β and H_α chemical shifts were (i + 1) < (i + 2). The distributions of the central two residues in type II and VIII β-turns were also distinguishable by trends of chemical shift values. Two-dimensional cluster analyses on chemical-shift data show positional distributions more clearly. Based on these propensities of chemical shift classified as a function of position, rules were derived using scoring matrices for four consecutive residues to predict type I and II β-turns. The proposed method achieves an overall prediction accuracy of 83.2 and 84.2 % with the Matthews correlation coefficient values of 0.317 and 0.632 for type I and II β-turns, indicating that its higher accuracy for type II turn prediction. The results show that it is feasible to use NMR chemical shifts to predict the β-turn types in proteins. The proposed method can be incorporated into other chemical-shift based protein secondary structure prediction methods.     

The effects of guanidine hydrochloride on the ’random coil‘ conformations and NMR chemical shifts of the peptide series GGXGG

The effects of the commonly used denaturant guanidine hydrochloride(GuHCl) on the random coil conformations and NMR chemical shifts of theproteogenic amino acids have been characterized using the peptide seriesAc-Gly-Gly-X-Gly-Gly-NH₂. The angle-sensitive couplingconstants, ROESY cross peak intensities and proline cis–trans isomerratios of a representative subset of these peptides are unaffected by GuHCl,which suggests that the denaturant does not significantly perturb intrinsicbackbone conformational preferences. A set of³J_HNH values is presented which agreewell with predictions of recently developed models of the random coil. Wehave also measured the chemical shifts of all 20 proteogenic amino acids inthese peptides over a range of GuHCl concentrations. The shifts exhibit alinear dependence on denaturant concentration and we report here correctionfactors for the calculation of random coil ¹H chemicalshifts at any arbitrary denaturant concentration. Studies of arepresentative subset of peptides indicate that ¹³C and¹⁵N chemical shifts are also perturbed by the denaturant.These results should facilitate the application of chemical shift-basedanalytical techniques to the study of polypeptides in solution with GuHCl.The effects of the denaturant on the quality of NMR spectra and on chemicalshift referencing are also addressed.     

The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-Xylosidase I

The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved β-Xylosidase and α-L: -Arabinofuranosidase (β-Xyl I-α-L: -Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant β-Xyl I-α-L: -Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant β-Xyl I-α-L: -Ara exhibited a specific β-Xylosidase I activity of 1.25?U?mg(-1) to oNPX and a specific α-L: -Arabinofuranosidase activity of 0.47?U?mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its β-Xylosidase I activity, and the enzymatic characterization was focused on it. The β-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45?°C, and a high degree of stability was verified over 240?min at this temperature. Moreover, β-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89?±?0.13?mM and 1.4?±?0.04?μM?min(-1) to oNPX, respectively. The modeled structure of β-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.     

NMR characterization of the interactions between lyso-GM1 aqueous micelles and amyloid β

Gangliosides are targets for a variety of pathologically relevant proteins, including amyloid β (Aβ), an important component implicated in Alzheimer’s disease (AD). To provide a structural basis for this pathogenic interaction associated with AD, we conducted NMR analyses of the Aβ interactions with gangliosides using lyso-GM1 micelles as a model system. Our NMR data revealed that the sugar-lipid interface is primarily perturbed upon binding of Aβ to the micelles, underscoring the importance of the inner part of the ganglioside cluster for accommodating Aβ in comparison with the outer carbohydrate branches that provide microbial toxin- and virus-binding sites.     

NMR structure of the transmembrane domain of the n-acetylcholine receptor β2 subunit

Nicotinic acetylcholine receptors (nAChRs) are involved in fast synaptic transmission in the central and peripheral nervous system. Among the many different types of subunits in nAChRs, the β2 subunit often combines with the α4 subunit to form α4β2 pentameric channels, the most abundant subtype of nAChRs in the brain. Besides computational predictions, there is limited experimental data available on the structure of the β2 subunit. Using high-resolution NMR spectroscopy, we solved the structure of the entire transmembrane domain (TM1234) of the β2 subunit. We found that TM1234 formed a four-helix bundle in the absence of the extracellular and intracellular domains. The structure exhibited many similarities to those previously determined for the Torpedo nAChR and the bacterial ion channel GLIC. We also assessed the influence of the fourth transmembrane helix (TM4) on the rest of the domain. Although secondary structures and tertiary arrangements were similar, the addition of TM4 caused dramatic changes in TM3 dynamics and subtle changes in TM1 and TM2. Taken together, this study suggests that the structures of the transmembrane domains of these proteins are largely shaped by determinants inherent in their sequence, but their dynamics may be sensitive to modulation by tertiary and quaternary contacts.     

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 plant Pentadiplandra 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 sideehain proton resonances were achieved using through-bond and through-space eonneetivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one α-helix (residues 21—29), one short 3_(10)-helix (residues 14—17), two strands of antiparallel β-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 'eysteine-stabilized alpha-beta' (CSαβ) motif with scorpion neurotoxins, insect defensins and plant γ-thionins. The significance of this multi-function motif, the possible active sites an     

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.     

The effects of cholesterol and β-sitosterol on the structure of saturated diacylphosphatidylcholine bilayers

The structures of DMPC and DPPC bilayers in unilamellar liposomes, in the presence of 33.3 mol% cholesterol or the plant sterol β-sitosterol, have been studied by small-angle neutron scattering. The bilayer thickness d _L increases in a similar way for both sterols. The repeat distance in multilamellar liposomes, as determined by small-angle X-ray diffraction, is larger in the presence of β-sitosterol than in the presence of cholesterol. We observe that each sterol modifies the interlamellar water layer differently, cholesterol reducing its thickness more efficiently than β-sitosterol, and conclude that cholesterol suppresses bilayer undulations more effectively than β-sitosterol.     

The Effect of pH and Heat Pre-Treatments on the Physicochemical and Emulsifying Properties of β-lactoglobulin

The overall goal of this research was to investigate structure-function mechanisms associated the emulsifying properties β-lactoglobulin (β-LG). Specifically the physicochemical (i.e., surface charge and hydrophobicity, size and interfacial tension) and emulsifying (i.e., emulsification activity (EAI) and stability indices (ESI)) properties of β-LG were investigated in response to changes in pH (3.0, 5.0 and 7.0) and heat pre-treatment conditions (25, 55 and 85 °C). Hydrophobicity was found to be greatest at pH 5.0/85 °C, whereas at all conditions it was significantly lower. Surface charge on β-LG was found to be neutral at?~?pH 3.9, regardless of conditions. Aggregate size was also found to be highest at pH 5.0/85 °C (avg. hydrodynamic radii of ~714 nm), corresponding to a reduced net surface charge and high hydrophobicity. Little size dependence of aggregates was observed at pH 3.0 regardless to the temperature pre-treatments (radii ~120 nm). In contrast, at pH 7.0 slight temperature dependence was apparent, where treatments at 25, 55 and 85 °C led to radii of 412.8, 307.2 and 232.3 nm, respectively. Overall, the addition of β-LG to a canola oil–water system resulted in a decline in interfacial tension from ~28 mN/m to ~18 mN/m, however the effect of pH/temperature conditions was minimal. EAI was found to be highest when β-LG solutions displayed high surface charge combined with moderate hydrophobicity. In contrast, ESI was higher under conditions where β-LG solutions remained in a native (25 °C) or fully denatured state (85 °C) versus one in where partially unravelling may be occurring (55 °C).     

Prediction of proton chemical shifts in RNA – Their use in structure refinement and validation

An analysis is presented of experimental versus calculated chemical shifts of the non-exchangeable protons for 28 RNA structures deposited in the Protein Data Bank, covering a wide range of structural building blocks. We have used existing models for ring-current and magnetic-anisotropy contributions to calculate the proton chemical shifts from the structures. Two different parameter sets were tried: (i) parameters derived by Ribas-Prado and Giessner-Prettre (GP set) [(1981) J. Mol. Struct., 76, 81–92.]; (ii) parameters derived by Case [(1995) J. Biomol. NMR, 6, 341–346]. Both sets lead to similar results. The detailed analysis was carried using the GP set. The root-mean-square-deviation between the predicted and observed chemical shifts of the complete database is 0.16 ppm with a Pearson correlation coefficient of 0.79. For protons in the usually well-defined A-helix environment these numbers are, 0.08 ppm and 0.96, respectively. As a result of this good correspondence, a reliable analysis could be made of the structural dependencies of the ¹H chemical shifts revealing their physical origin. For example, a down-field shift of either H2 or H3 or both indicates a high-syn/syn -angle. In an A-helix it is essentially the 5-neighbor that affects the chemical shifts of H5, H6 and H8 protons. The H5, H6 and H8 resonances can therefore be assigned in an A-helix on the basis of their observed chemical shifts. In general, the chemical shifts were found to be quite sensitive to structural changes. We therefore propose that a comparison between calculated and observed ¹H chemical shifts is a good tool for validation and refinement of structures derived from NOEs and J-couplings.     

Comparative analysis of 13C chemical shifts of β-sheet amyloid proteins and outer membrane proteins

Cross-β amyloid fibrils and membrane-bound β-barrels are two important classes of β-sheet proteins. To investigate whether there are systematic differences in the backbone and sidechain conformations of these two families of proteins, here we analyze the ¹³C chemical shifts of 17 amyloid proteins and 7 β-barrel membrane proteins whose high-resolution structures have been determined by NMR. These 24 proteins contain 373 β-sheet residues in amyloid fibrils and 521 β-sheet residues in β-barrel membrane proteins. The ¹³C chemical shifts are shown in 2D ¹³C–¹³C correlation maps, and the amino acid residues are categorized by two criteria: (1) whether they occur in β-strand segments or in loops and turns; (2) whether they are water-exposed or dry, facing other residues or lipids. We also examine the abundance of each amino acid in amyloid proteins and β-barrels and compare the sidechain rotameric populations. The ¹³C chemical shifts indicate that hydrophobic methyl-rich residues and aromatic residues exhibit larger static sidechain conformational disorder in amyloid fibrils than in β-barrels. In comparison, hydroxyl- and amide-containing polar residues have more ordered sidechains and more ordered backbones in amyloid fibrils than in β-barrels. These trends can be explained by steric zipper interactions between β-sheet planes in cross-β fibrils, and by the interactions of β-barrel residues with lipid and water in the membrane. These conformational trends should be useful for structural analysis of amyloid fibrils and β-barrels based principally on NMR chemical shifts.

     

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.     

Isolation and characterization of β-d-glucan,heteropolysaccharide, and trehalose components of the basidiomycetous lichen Cora pavonia

Cora pavonia, a lichen having a basidiomycetous mycobiont, is rich in protein (36%), of which 10% is tyrosine. α,α-Trehalose is present and was isolated in 4.4% yield. The lichen was found to contain polysaccharides typical of basidiomycetes and different from those of ascomycetes and ascomycetous lichens. Isolated and characterized were a β-d-glucan and a heteropolysaccharide containing l-rhamnose, l-fucose, d-xylose, d-mannose, d-glucose, and d-galactose. The β-d-glucan was highly branched with 21% of nonreducing end-groups, contained 3-O-, 6-O-, and 3,6-di-O-substituted β-d-glucopyranosyl units, and had a main chain consisting of (1→3)- and (1→6)-links. The heteropolysaccharide component contained mainly mannose and xylose, having a mannose-containing nucleus and a main chain with preponderant (1→3)-linked α-d-mannopyranosyl residues. These were unsubstituted (10%), and 4-O- (10%) and 2,4-di-O-substituted (10%) with residues of β-d-xylopyranose. On methylation analysis of the heteropolysaccharide, a capillary column of DB-210 proved to be particularly useful for gas-liquid chromatographic resolution of partially O-methylated alditol acetates.     

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.