Efficient recovery of glycosaminoglycan oligosaccharides from polyacrylamide gel electrophoresis combined with mass spectrometry analysis.

To promote efficient separation and structural analysis of glycosaminoglycan oligosaccharides, we developed a straightforward method that combined gel electrophoresis and mass spectrometry (MS). Potential limitations of this approach (e.g., low extraction yields and weak compatibility with MS) were resolved by developing an active extraction procedure that yielded a quantitative amount of sulfated oligosaccharides from excised gel bands. The compatibility of obtained oligosaccharides for subsequent MS analysis was ensured using a single, simple clean-up step on a mixed C18/graphite carbon solid-phase column that was fully effective for polymerization degrees ranging from di- to dodecasaccharides. The reported combination of carbohydrates-polyacrylamide gel electrophoresis with MS was successfully applied to glucosamino- (heparin) and galactosamino- (dermantan sulfate) glycans, demonstrating the potential of our method for structural analysis of bioactive sulfated carbohydrates extracted from biological matrices. Graphical Abstract ᅟ.

Probing the solution structure of Factor H using hydroxyl radical protein footprinting and cross-linking.

The control protein Factor H (FH) is a crucial regulator of the innate immune complement system, where it is active on host cell membranes and in the fluid phase. Mutations impairing the binding capacity of FH lead to severe autoimmune diseases. Here, we studied the solution structure of full-length FH, in its free state and bound to the C3b complement protein. To do so, we used two powerful techniques, hydroxyl radical protein footprinting (HRPF) and chemical cross-linking coupled with mass spectrometry (MS), to probe the structural rearrangements and to identify protein interfaces. The footprint of C3b on the FH surface matches existing crystal structures of C3b complexed with the N- and C-terminal fragments of FH. In addition, we revealed the position of the central portion of FH in the protein complex. Moreover, cross-linking studies confirmed the involvement of the C-terminus in the dimerization of FH.

Understanding the mechanism of short-range electron transfer using an immobilized cupredoxin.

The hydrophobic patch of azurin (AZ) from Pseudomonas aeruginosa is an important recognition surface for electron transfer (ET) reactions. The influence of changing the size of this region, by mutating the C-terminal copper-binding loop, on the ET reactivity of AZ adsorbed on gold electrodes modified with alkanethiol self-assembled monolayers (SAMs) has been studied. The distance-dependence of ET kinetics measured by cyclic voltammetry using SAMs of variable chain length, demonstrates that the activation barrier for short-range ET is dominated by the dynamics of molecular rearrangements accompanying ET at the AZ-SAM interface. These include internal electric field-dependent low-amplitude protein motions and the reorganization of interfacial water molecules, but not protein reorientation. Interfacial molecular dynamics also control the kinetics of short-range ET for electrostatically and covalently immobilized cytochrome c. This mechanism therefore may be utilized for short-distance ET irrespective of the type of metal center, the surface electrostatic potential, and the nature of the protein-SAM interaction.

Metal-binding loop length and not sequence dictates structure.

The C-terminal copper-binding loop in the beta-barrel fold of the cupredoxin azurin has been replaced with a range of sequences containing alanine, glycine, and valine residues to assess the importance of amino acid composition and the length of this region. The introduction of 2 and 4 alanines between the coordinating Cys, His, and Met results in loop structures matching those in naturally occurring proteins with the same loop lengths. A loop with 4 alanines between the Cys and His and 3 between the His and Met ligands has a structure identical to that of the WT protein, whose loop is the same length. Loop structure is dictated by length and not sequence allowing the properties of the main surface patch for interactions with partners, to which the loop is a major contributor, to be optimized. Loops with 2 amino acids between the ligands using glycine, alanine, and valine residues have been compared. An empirical relationship is found between copper site protection by the loop and reduction potential. A loop adorned with 4 methyl groups is sufficient to protect the copper ion, enabling most sequences to adequately perform this task. The mutant with 3 alanine residues between the ligands forms a strand-swapped dimer in the crystal structure, an arrangement that has not, to our knowledge, been seen previously for this family of proteins. Cupredoxins function as redox shuttles and are required to be monomeric; therefore, none have evolved with a metal-binding loop of this length.

A new cytochrome P450 belonging to the 107L subfamily is responsible for the efficient hydroxylation of the drug terfenadine by Streptomyces platensis.

Fexofenadine, an antihistamine drug used in allergic rhinitis treatment, can be produced by oxidative biotransformation of terfenadine by Streptomyces platensis, which involves three consecutive oxidation reactions. We report here the purification and identification of the enzyme responsible for the first step, a cytochrome P450 (P450)-dependent monooxygenase. The corresponding P450, designated P450(terf), was found to catalyze the hydroxylation of the t-butyl group of terfenadine and exhibited UV-Vis characteristics of a P450. Its interaction with terfenadine led to a shift of its Soret peak from 418 to 390 nm, as expected for the formation of a P450-substrate complex. In combination with spinach ferredoxin:NADP(+) oxidoreductase and ferredoxin, and in the presence of NADPH, it catalyzed the hydroxylation of terfenadine and some of its analogues, such as terfenadone and ebastine, with k(m) values at the µM level, and k(cat) values around 30min(-1). Sequencing of the p450(terf) gene led to a 1206 bp sequence, encoding for a 402 aminoacid polypeptide exhibiting 56-65% identity with the P450s from the 107L family. These results confirmed that P450s from Streptomyces species are interesting tools for the biotechnological production of secondary metabolites, such as antibiotics or antitumor compounds, and in the oxidative biotransformation of xenobiotics, such as drugs.

Metal-binding loop length is a determinant of the pKa of a histidine ligand at a type 1 copper site.

The type 1 copper site of a cupredoxin involves coordination by cysteine, histidine, and methionine residues from a single loop. Dissociation and protonation of the histidine ligand on this loop is observed in only certain reduced cupredoxins and can regulate electron-transfer reactivity. This effect is introduced in azurin (AZ) (the wild-type protein has an estimated pKa of <2) by mutating the native copper-binding loop (C(112)TFPGH(117)SALM(121), ligands numbered). In this work, we have investigated the influence of loop length alone on histidine ligand protonation by determining the pKa value in AZ variants with ligand-containing polyalanine loops of different length. Crystal structures of the Cu(I)-variant with the loop sequence C(112)AAH(115)AAM(118) (AZ2A2A) demonstrate that at pH 4.2 His115 is protonated and no longer coordinated, and the imidazole ring is rotated by 180°. The influence of pH on the reduction potential allows a pKa of 5.2 ± 0.1 for His115 in Cu(I)-AZ2A2A to be determined. In the reduced AZ variants in which the loop sequences C(112)AAAAH(117)AAAM(121) (AZ4A3A) and C(112)AAAAH(117)AAAAM(122) (AZ4A4A) have been introduced, pKa values of 4.5 ± 0.1 and 4.4 ± 0.1, respectively, are obtained for the His117 ligand. Consistent with these data, the crystal structure of Cu(I)-AZ4A4A at pH 5.3 shows no sign of His117 protonation (crystals were unstable at lower pH values). The loop length range studied matches that which occurs naturally and these investigations indicate that length alone can alter the pKa of the coordinating histidine by approximately 1 pH unit. The pKa for this histidine ligand varies in native cupredoxins by >5 pH units. Other structural and electronic features, governed primarily by the second-coordination sphere, to which the ligand-binding loop is a major contributor, also alter this important feature. A longer ligand-containing loop made of residues whose side chains are larger and more complex than a methyl group increases the second coordination sphere providing additional scope for tuning the pKa of the histidine ligand and other active site properties.

Analogies and surprising differences between recombinant nitric oxide synthase-like proteins from Staphylococcus aureus and Bacillus anthracis in their interactions with l-arginine analogs and iron ligands.

Genome sequencing has recently shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles of these proteins remain unclear. The interactions of a series of l-arginine (l-arg) analogs and iron ligands with two recombinant NOS-like proteins from Staphylococcus aureus (saNOS) and Bacillus anthracis (baNOS) have been studied by UV-visible spectroscopy. SaNOS and baNOS in their ferric native state, as well as their complexes with l-arg analogs and with various ligands, exhibit spectral characteristics highly similar to the corresponding complexes of heme-thiolate proteins such as cytochromes P450 and NOSs. However, saNOS greatly differs from baNOS at the level of three main properties: (i) native saNOS mainly exists under an hexacoordinated low-spin ferric state whereas native baNOS is mainly high-spin, (ii) the addition of tetrahydrobiopterin (H4B) or H4B analogs leads to an increase of the affinity of l-arg for saNOS but not for baNOS, and (iii) saNOS Fe(II), contrary to baNOS, binds relatively bulky ligands such as nitrosoalkanes and tert-butylisocyanide. Thus, saNOS exhibits properties very similar to those of the oxygenase domain of inducible NOS (iNOS(oxy)) not containing H4B, as expected for a NOSoxy-like protein that does not contain H4B. By contrast, the properties of baNOS which look like those of H4B-containing iNOS(oxy) are unexpected for a NOS-like protein not containing H4B. The origin of these surprising properties of baNOS remains to be determined.