Structural characterization of Escherichia coli BamE, a lipoprotein component of the β-barrel assembly machinery complex

In Escherichia coli, the BAM complex catalyzes the essential process of assembling outer membrane proteins (OMPs). This complex consists of five proteins: one membrane-bound protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their importance in OMP biogenesis, there is currently a lack of functional and structural information on the BAM complex lipoproteins. BamE is the smallest but most conserved lipoprotein in the complex. The structural and dynamic properties of monomeric BamE (residues 21-133) were determined by NMR spectroscopy. The protein folds as two α-helices packed against a three-stranded antiparallel β-sheet. The N-terminal (Ser21-Thr39) and C-terminal (Pro108-Asn113) residues, as well as a β-hairpin loop (Val76-Gln89), are highly flexible on the subnanosecond time scale. BamE expressed and purified from E. coli also exists in a kinetically trapped dimeric state that has dramatically different NMR spectra, and hence structural features, relative to its monomeric form. The functional significance of the BamE dimer remains to be established. Structural comparison to proteins with a similar architecture suggests that BamE may play a role in mediating the association of the BAM complex or with the BAM complex substrates.     

Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of ¹H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond ¹H–¹⁵N or ¹H–¹³C couplings. However, by filtering against protons directly bonded to ¹³C or ¹⁵N nuclei, signals from slowly-exchanging hydroxyls can be observed in the ¹H-NMR spectrum of a uniformly ¹³C/¹⁵N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with ¹³C/¹⁵N-serine or ¹³C/¹⁵N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via ³J_C–OH couplings in long-range ¹³C-HSQC spectra. These scalar couplings (~5–7 Hz) were measured in a sample of uniformly ¹³C/¹⁵N-labeled BcX using a quantitative ¹³C/¹⁵N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a ¹³C/¹⁵N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.     

pH-dependent random coil 1H, 13C,and 15N chemical shifts of the ionizable amino acids: a guide for protein pK a measurements

The pK _a values and charge states of ionizable residues in polypeptides and proteins are frequently determined via NMR-monitored pH titrations. To aid the interpretation of the resulting titration data, we have measured the pH-dependent chemical shifts of nearly all the ¹H, ¹³C, and ¹⁵N nuclei in the seven common ionizable amino acids (X = Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, respectively. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor reference chemical shifts to help guide protein pK _a measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chemical shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain ¹⁵N nuclei of His, Lys, and Arg in their positively-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary experimental evidence for defining the ionization states of protein residues.     

Structure-Function Analysis of a Broad Specificity Populus trichocarpa Endo-β-glucanase Reveals an Evolutionary Link between Bacterial Licheninases and Plant XTH Gene Products

The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/β-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage β(1→3)/β(1→4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins.     

Arginine: Its pKa value revisited

Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pK_a value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pK_a value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pK_a value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group.     

Theoretical and experimental studies on the inhibition potentials of aromatic oxaldehydes for the corrosion of mild steel in 0.1 M HCl

Experimental aspect of the inhibition of the corrosion of mild steel by oxaldehydes was carried out using gravimetric, gasometric and thermometric methods while the theoretical studies were carried out using quantum chemical principle and quantitative structure activity relation (QSAR) approaches. The results obtained indicated that the studied oxaldehydes are good inhibitors for the corrosion of mild steel in HCl solutions. The adsorption of the inhibitors on mild steel surface is spontaneous, exothermic and is consistent with the assumptions of Langmuir adsorption isotherm. Excellent correlations were found between the calculated quantum chemical parameters and experimental inhibition efficiencies of the studied compounds. Correlations between theoretical and experimental inhibition efficiencies (for the different Hamiltonians, namely, PM6, PM3, AM1, RM1 and MNDO) were very close to unity. Condensed Fukui function and condensed softness have been used to determine the sites for electrophilic and nucleophilic attacks on each of the inhibitors.     

The Vicia sativa spp. nigra - Rhizobium leguminosarum bv. viciae symbiotic interaction is improved by Azospirillum brasilense

This study aimed to examine the effects of inoculation with wild type (Sp7) and mutant strains of Azospirillum brasilense on the Vicia sativa spp. nigra (vetch)-Rhizobium leguminosarum bv. viciae (Rlv) symbiosis. The A. brasilense mutants were ipdC ^- and napA ^-, impaired in indole pyruvate decarboxylase and periplasmic nitrate reductase, respectively; and acdS ⁺ , carrying the ACC deaminase gene. Inoculations were done in pots, pouches and hydroponics and we measured shoot and root weight parameters as well as effects on root morphology and nod gene induction ability by roots. In pots, wild type Sp7 and the acdS ⁺ strain, but ipdC ^- and napA ^- mutants, lead to an increase in root hair density, 3–4?cm above the root tip. In pouches, combined inoculation with Rlv and strains Sp7, acdS ⁺ or ipdC ^-, but napA ^-, increased shoot dry matter and nodulation relative to Rlv alone. In a hydroponic system, co-inoculation with strains Sp7 or acdS ⁺ , but with ipdC ^- and napA ^- mutants, enhanced root secretion of nod gene-inducing flavonoids in comparison with Rlv-inoculated plants. These results support that auxin production by A. brasilense has a positive effect on root secretion of nod gene-inducing flavonoids and auxin absorption activity by the plant.     

Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation

The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.