Complexation and synergistic boundary lubrication of porcine gastric mucin and branched poly(ethyleneimine) in neutral aqueous solution.

Lubrication of soft polydimethylsiloxane (PDMS) elastomer interfaces was studied in aqueous mixtures of porcine gastric mucin (PGM) and branched polyethyleneimine (b-PEI) at neutral pH and various ionic strengths (0.1-1.0 M). While neither PGM nor b-PEI improved lubrication compared to polymer-free buffer solution, their mixtures produced a synergistic lubricating effect by reducing friction coefficients by nearly two orders of magnitude, especially at slow sliding speed in the boundary lubrication regime. An array of spectroscopic studies revealed that small cationic b-PEI molecules were able to strongly bind and penetrate into large anionic PGM molecules, producing an overall contraction of the randomly coiled PGM conformation. The interaction also affected the structure of the folded PGM protein terminals, decreased the surface potential and increased light absorbance in PGM:b-PEI mixtures. Adding an electrolyte (NaCl) weakened the aggregation between PGM and b-PEI, and degraded the lubrication synergy, indicating that electrostatic interactions drive PGM:b-PEI complexation.

Variations of subunit {varepsilon} of the Mycobacterium tuberculosis F1Fo ATP synthase and a novel model for mechanism of action of the tuberculosis drug TMC207.

The subunit ε of bacterial F(1)F(O) ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of ε of Mycobacterium tuberculosis (Mtε) F(1)F(O) ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment (Mtε(103-120)). Mtε is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic ß subunit. The C-terminal segment displays an α-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c-ring, also binds to Mtε. A model for the interaction of TMC207 with both ε and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of ε and the c-ring, respectively. T19 and R37 of ε provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F(1)F(O) ATP synthase in M. tuberculosis.

The NMR solution structure of subunit G (G(61)(-)(101)) of the eukaryotic V1VO ATPase from Saccharomyces cerevisiae.

Subunit G is an essential stalk subunit of the eukaryotic proton pump V(1)V(O) ATPase. Previously the structure of the N-terminal region, G(1)(-)(59), of the 13kDa subunit G was solved at higher resolution. Here solution NMR was performed to determine the structure of the recombinant C-terminal region (G(61)(-)(101)) of subunit G of the Saccharomyces cerevisiae V(1)V(O) ATPase. The protein forms an extended alpha-helix between residues 64 and 100, whereby the first five- and the last residues of G(61)(-)(101) are flexible. The surface charge distribution of G(61)(-)(101) reveals an amphiphilic character at the C-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The hydrophobic surface pattern is mainly formed by alanine residues. The alanine residues 72, 74 and 81 were exchanged by a single cysteine in the entire subunit G. Cysteines at positions 72 and 81 showed disulfide formation. In contrast, no crosslink could be formed for the mutant Ala74Cys. Together with the recently determined NMR solution structure of G(1)(-)(59), the presented solution structure of G(61)(-)(101) enabled us to present a first structural model of the entire subunit G of the S. cerevisiae V(1)V(O) ATPase.

Structural elements of the C-terminal domain of subunit E (E133₋222) from the Saccharomyces cerevisiae V1V0 ATPase determined by solution NMR spectroscopy.

Subunit E of the vacuolar ATPase (V-ATPase) contains an N-terminal extended α helix (Rishikesan et al. J Bioenerg Biomembr 43:187-193, 2011) and a globular C-terminal part that is predicted to consist of a mixture of α-helices and ß-sheets (Grüber et al. Biochem Biophys Res Comm 298:383-391, 2002). Here we describe the production, purification and 2D structure of the C-terminal segment E133₋222 of subunit E from Saccharamyces cerevisiae V-ATPase in solution based on the secondary structure calculation from NMR spectroscopy studies. E133₋222 consists of four ß-strands, formed by the amino acids from K136-V139, E170-V173, G186-V189, D195-E198 and two α-helices, composed of the residues from R144-A164 and T202-I218. The sheets and helices are arranged as ß1:α1:ß2:ß3:ß4:α2, which are connected by flexible loop regions. These new structural details of subunit E are discussed in the light of the structural arrangements of this subunit inside the V1- and V1V0 ATPase.

NMR solution structure of subunit E (fragment E(1-69)) of the Saccharomyces cerevisiae V (1)V (O) ATPase.

The N-terminus of V-ATPase subunit E has been shown to associate with the subunits C, G and H, respectively. To understand the assembly of E with its neighboring subunits as well as its N-terminal structure, the N-terminal region, E(1-69), of the Saccharomyces cerevisiae V-ATPase subunit E was expressed and purified. The solution structure of E(1-69) was determined by NMR spectroscopy. The protein is 90.3 Å in length and forms an á-helix between the residues 12-68. The molecule is amphipathic with hydrophobic residues at the N-terminus, predicted to interact with subunit C. The polar epitopes of E(1-69) are discussed as areas interacting with subunits G and H.

Mycobacterium tuberculosis PanD Structure-Function Analysis and Identification of a Potent Pyrazinoic Acid-Derived Enzyme Inhibitor.

A common strategy employed in antibacterial drug discovery is the targeting of biosynthetic processes that are essential and specific for the pathogen. Specificity in particular avoids undesirable interactions with potential enzymatic counterparts in the human host, and it ensures on-target toxicity. Synthesis of pantothenate (Vitamine B5), which is a precursor of the acyl carrier coenzyme A, is an example of such a pathway. In Mycobacterium tuberculosis (Mtb), which is the causative agent of tuberculosis (TB), pantothenate is formed by pantothenate synthase, utilizing D-pantoate and ß-Ala as substrates. ß-Ala is mainly formed by the decarboxylation of l-aspartate, generated by the decarboxylase PanD, which is a homo-oliogomer in solution. Pyrazinoic acid (POA), which is the bioactive form of the TB prodrug pyrazinamide, binds and inhibits PanD activity weakly. Here, we generated a library of recombinant Mtb PanD mutants based on structural information and PZA/POA resistance mutants. Alterations in oligomer formation, enzyme activity, and/or POA binding were observed in respective mutants, providing insights into essential amino acids for Mtb PanD's proper structural assembly, decarboxylation activity and drug interaction. This information provided the platform for the design of novel POA analogues with modifications at position 3 of the pyrazine ring. Analogue 2, which incorporates a bulky naphthamido group at this position, displayed a 1000-fold increase in enzyme inhibition, compared to POA, along with moderately improved antimycobacterial activity. The data demonstrate that an improved understanding of mechanistic and enzymatic features of key metabolic enzymes can stimulate design of more-potent PanD inhibitors.

Assembly of subunit d (Vma6p) and G (Vma10p) and the NMR solution structure of subunit G (G(1-59)) of the Saccharomyces cerevisiae V(1)V(O) ATPase.

Understanding the structural traits of subunit G is essential, as it is needed for V(1)V(O) assembly and function. Here solution NMR of the recombinant N- (G(1-59)) and C-terminal segment (G(61-114)) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G(1-59) only. The residues of G(1-59) involved in d binding are Gly7 to Lys34. The structure of G(1-59) has been solved, revealing an alpha-helix between residues 10 and 56, whereby the first nine- and the last three residues of G(1-59) are flexible. The surface charge distribution of G(1-59) reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G(1-59)-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E(18-38) of subunit E of yeast V-ATPase and the presently solved structure of G(1-59), both proteins have been docked and binding epitopes have been analyzed.

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E.

A critical point in the V(1) sector and entire V(1)V(O) complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G(1-59)) of the V(1)V(O) ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of G(1-59) in the absence and presence of the N-terminal peptides E(1-18) and E(18-38) as well as the produced and purified C-terminal segment (E(39-233)) shows specific interactions only with the peptide fragment E(18-38). The binding of this peptide occurs via the residues M(1), V(2), S(3), and K(5) as well for V(22), S(23), K(24), A(25) and R(26) of G(1-59). The specific E(18-38)/G(1-59) binding has been confirmed by fluorescence correlation spectroscopy data. The E(18-38) peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E(18-38)/G(1-59) complex reveals the orientation of E(18-38) relative to G(1-59) via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.

Gastric mucus and mucuslike hydrogels: Thin film lubricating properties at soft interfaces.

Mucus is a viscous slime that plays a vital role in protecting and lubricating biological tissues, in particular, soft epithelium interfaces such as in the stomach, intestines, and esophagus. Previous attempts to generate mucus models that mimick or simulate its characteristics have been predominantly focused on the rheological properties. This study investigates both rheological and tribological shear properties of thin films of gastric mucus from a porcine source and its mimics at compliant soft interfaces. The lubricating efficacy of biological mucus and its mimics was observed to be superior at hydrophilic tribological interfaces compared to hydrophobic ones. Facile spreading of all mucus samples at hydrophilic steel-polydimethylsiloxane (PDMS) interfaces allowed for the retainment of the lubricating films over a wide range of speed, slide/roll ratio, and external load. In contrast, poor wetting at hydrophobic PDMS-PDMS interfaces led to depletion of the mucus samples from the interface with increasing speed. Among the different mucus models investigated in this study, fluid mixtures of commercially available porcine gastric mucin (PGM) and polyacrylic acid (PAA) displayed the most persistent lubricating effects under various tribological experimental conditions. A mixture of PGM and PAA holds a high potential as mucus mimic, not only for its rheological similarity, but also for its excellent lubricity in soft compliant and hydrophilic contacts.

Slippery when sticky: Lubricating properties of thin films of Taxus baccata aril mucilage.

Mucilage is hydrogel produced from succulent plants and microorganisms displaying unique adhesiveness and slipperiness simultaneously. The objective of this study is to establish an understanding on the lubricating mechanisms of the mucilage from Taxus baccata aril as thin, viscous lubricant films. Oscillation and flow rheological studies revealed that T. baccata mucilage is shear-thinning, thixotropic, and weak hydrogel that is highly stretchable under shear stress due to its high density physical crosslinking characteristics. In addition, T. baccata mucilage showed a distinct Weissenberg effect, i.e., increasing normal force with increasing shear rate, and thus it contributes to deplete the lubricant from tribological interfaces. Lubrication studies with a number of tribopairs with varying mechanical properties and surface wettability have shown that the lubricity of T. baccata mucilage is most effectively manifested at soft, hydrophilic, and rolling tribological contacts. Based on tenacious spreading on highly wetting surfaces, slip plane can be formed within mucilage hydrogel network even when the lubricating films cannot completely separate the opposing surfaces. Moreover, highly stretchable characteristics of mucilage under high shear enhance smooth shearing of two opposing surfaces as lubricating film.