The role of the key catalytic residues Glu134 and Glu138 in the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis is probed by a chemical rescue methodology based on enzyme activation of inactive mutants by the action of added nucleophiles. While Glu134 was proposed as the catalytic nucleophile on the basis of affinity labeling experiments, no functional proof supported the assignment of Glu138 as the general acid-base catalyst. Alanine replacements are prepared by site-directed mutagenesis to produce the inactive E138A and E134A mutants. Addition of azide reactivates the mutants in a concentration-dependent manner using an activated 2, 4-dinitrophenyl glycoside substrate. The chemical rescue operates by a different mechanism depending on the mutant as deduced from 1H NMR monitoring and kinetic analysis of enzyme reactivation. E138A yields the beta-glycosyl azide product arising from nucleophilic attack of azide on the glycosyl-enzyme intermediate, thus proving that Glu138 is the general acid-base residue. Azide activates the deglycosylation step (increasing kcat), but it also has a large effect on a previous step (as seen by the large decrease in KM, the increase in kcat/KM, and the pH dependence of activation), probably increasing the rate of glycosylation through Bronsted acid catalysis by enzyme-bound HN3. By contrast, azide reactivates the E134A mutant through a single inverting displacement to give the alpha-glycosyl azide product, consistent with Glu134 being the catalytic nucleophile. Formate as an exogenous nucleophile has no effect on the E138A mutant, whereas it is a better activator of E134A than azide. Although the reaction yields the normal hydrolysis product, a transient compound was detected by 1H NMR, tentatively assigned to the alpha-glycosyl formate adduct. This is the first case where a nonmodified sugar gives a long-lived covalent intermediate that mimics the proposed glycosyl-enzyme intermediate of retaining glycosidases.

Background: Mutations at Gly-90 in rhodopsin cause two different phenotypes: retinitis pigmentosa and congenital night blindness. Results: G90V retinitis pigmentosa mutant shows constitutive activity and very low thermal stability in the dark state. Conclusion: Low conformational stability can trigger retinitis pigmentosa associated with rhodopsin mutations. Significance: Retinoids can help to stabilize the conformation of retinitis pigmentosa mutants.

Drug resistance occurrence is a global healthcare concern responsible for the increased morbidity and mortality in hospitals, time of hospitalisation and huge financial loss. The failure of the most antibiotics to kill "superbugs" poses the urgent need to develop innovative strategies aimed at not only controlling bacterial infection but also the spread of resistance. The prevention of pathogen host invasion by inhibiting bacterial virulence and biofilm formation, and the utilisation of bactericidal agents with different mode of action than classic antibiotics are the two most promising new alternative strategies to overcome antibiotic resistance. Based on these novel approaches, researchers are developing different advanced materials (nanoparticles, hydrogels and surface coatings) with novel antimicrobial properties. In this review, we summarise the recent advances in terms of engineered materials to prevent bacteria-resistant infections according to the antimicrobial strategies underlying their design.

Heterotrimeric G-protein activation by a G-protein-coupled receptor (GPCR) requires the propagation of structural signals from the receptor-interacting surfaces to the guanine nucleotide-binding pocket. To probe conformational changes in the G-protein alpha-subunit (G(alpha)) associated with activated GPCR (R*) interactions and guanine nucleotide exchange, high-resolution solution NMR methods are being applied in studying signaling of the G-protein, transducin, by light-activated rhodopsin. Using these methods, we recently demonstrated that an isotope-labeled G(alpha) reconstituted heterotrimer forms functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin and a soluble mimic of R*, both of which trigger guanine nucleotide exchange [Ridge, K. D., et al. (2006) J. Biol. Chem. 281, 7635-7648]. Here, it is shown that both light-activated rhodopsin and the soluble mimic of R form trapped intermediate complexes with a GDP-released "empty pocket" state of the heterotrimer in the absence of GTP (or GTPgammaS). In contrast to guanine nucleotide-bound forms of G(alpha), the NMR spectra of the GDP-released, R-bound empty pocket state of G(alpha) display severe line broadening suggestive of a dynamic intermediate state. Interestingly, the conformation of a GDP-depleted, Mg(2+)-bound state of G(alpha) generated in a manner independent of R* does not exhibit a similar degree of line broadening but rather appears structurally similar to the GDP/Mg(2+)-bound form of the protein. Taken together, these results suggest that R*-mediated changes in the receptor-interacting regions of G(alpha), and not the absence of bound guanine nucleotide, are the predominant factors which dictate G(alpha) conformation and dynamics in this R*-bound state of the heterotrimer.

Zinc is present at high concentrations in the photoreceptor cells of the retina where it has been proposed to play a role in the visual phototransduction process. In order to obtain more information about this role, the study of the effect of zinc on several properties of the visual photoreceptor rhodopsin has been investigated. A specific effect of Zn 2؉ on the thermal stability of rhodopsin, obtained from bovine retinas and solubilized in dodecyl maltoside detergent, in the dark is reported. Rhodopsin is the photoreceptor protein of the vertebrate retina (1-3) belonging to the G-protein-coupled receptor (GPCR) 1 superfamily (4 -6). It is the main protein component of the rod outer segments (ROS) of the retinal photoreceptor cells, and its easy isolation from bovine retinas has made of this receptor a widely used model for the GPCR superfamily. Rhodopsin is a key molecule in the biochemistry of vision and alterations in its sequence have been associated with retinal disease. In particular, a high number of mutations in the opsin gene have been associated with the autosomal form of the retinal degenerative disease retinitis pigmentosa (7,8). A number of factors have been proposed to be related to retinal function, and among them a possible role for Zn 2ϩ in the retina and its metabolism has been proposed (9). Zn 2ϩ is present at particularly high concentrations in the retina (10) being a component of the disc membranes in the rod outer segments of the photoreceptor cells (11). Zn 2ϩ has been histochemically localized to ROS (12) and it has been shown to copurify with ROS proteins as well (13). Despite this presence, the role of Zn 2ϩ in the visual cycle and specifically in its interaction with rhodopsin remains unclear.Zn 2ϩ is required for the function of numerous proteins, serving both as a part of the active site in, for example, metalloenzymes, and acting to stabilize protein domains, such as the Zn 2ϩ fingerbinding motif in transcription factors (14, 15). The structure of many Zn 2ϩ -binding sites is known from x-ray crystallography of Zn 2ϩ -binding proteins and, thus, the geometry of the interaction between Zn 2ϩ and different coordinating residues is well characterized (14, 15). This, together with the small size of the zinc (II) ion, makes artificially generated Zn 2ϩ -binding sites a highly useful approach for probing structure-function relationships in proteins. In GPCRs, for example, construction of bis-His Zn 2ϩ -binding sites has led to important information about both the organization of the transmembrane helices and their movements during receptor activation to be obtained (16 -22). Therefore, engineered Zn 2ϩ binding sites created by site-directed mutagenesis is an interesting tool for probing intramolecular interactions in these types of receptors (23). Particularly in the case of rhodopsin, important structural information about helical orientation, connectivities, and the conformational change upon light activation, has been obtained from engineered Zn 2ϩ -binding sites in this visual photore...

Background:Two new rhodopsin mutations associated with the rare form sector retinitis pigmentosa (RP) have been found. Results: Characterization of both rhodopsin mutant proteins shows different progression correlating with a different behavior of rhodopsin upon light exposure. Conclusion: Light plays an important role in triggering sector RP. Significance: Other mechanisms, in addition to protein misfolding, underlie GPCR dysfunction in pathological processes.

Naturally occurring point mutations in the opsin gene cause the retinal diseases retinitis pigmentosa and congenital night blindness. Although these diseases involve similar mutations in very close locations in rhodopsin, their progression is very different, with retinitis pigmentosa being severe and causing retinal degeneration. We report on the expression and characterization of the recently found T94I mutation associated with congenital night blindness, in the second transmembrane helix or rhodopsin, and mutations at the same site. T94I mutant rhodopsin folded properly and was able to bind 11-cis-retinal to form chromophore, but it showed a blue-shifted visible band at 478 nm and reduced molar extinction coefficient. Furthermore, T94I showed dramatically reduced thermal stability, extremely long lived metarhodopsin II intermediate, and highly increased reactivity toward hydroxylamine in the dark, when compared with wild type rhodopsin. The results are consistent with the location of Thr-94 in close proximity to Glu-113 counterion in the vicinity of the Schiff base linkage and suggest a role for this residue in maintaining the correct dark inactive conformation of the receptor. The reported results, together with previously published data on the other two known congenital night blindness mutants, suggest that the molecular mechanism underlying this disease may not be structural misfolding, as proposed for retinitis pigmentosa mutants, but abnormal functioning of the receptor by decreased thermal stability and/or constitutive activity.Naturally occurring mutations in rhodopsin (most of them single amino acid replacements) are associated with retinal disease. Most of these are the cause of retinitis pigmentosa (RP), 1 a group of inherited retinal degenerative diseases (1-3) that leads to blindness by causing photoreceptor cell death (4). Over 100 mutations have been found to date in the opsin gene associated with RP, most of them being inherited as an autosomal dominant trait (1). These are located in all the three domains of rhodopsin, namely the intradiscal, the transmembrane, and the cytoplasmic domains of the protein (5). Mutations in the transmembrane and intradiscal domains of rhodopsin that cause RP have been shown to cause misfolding of the mutant proteins (6 -8). Only a very small number of mutations have been associated with the retinal disease characterized by a congenital night blindness (CNB) phenotype. CNB appears to be a stable condition that does not seem to cause photoreceptor degeneration resulting mainly in night vision impairment. Two of these mutations were previously studied, namely G90D (9) and A292E (10) in transmembrane helices II and VII of rhodopsin, respectively. The mechanism of action of the G90D and A292E mutations was proposed to be persistent activation of the phototransduction pathway by constitutive activity of the mutant proteins (9 -11). Another possible explanation for the observed G90D mutant phenotype has been proposed, i.e. enhanced rate of thermal isomerization due to lo...

Dietary flavonoids exhibit many biologically-relevant functions and can potentially have beneficial effects in the treatment of pathological conditions. In spite of its well known antioxidant properties, scarce structural information is available on the interaction of flavonoids with membrane receptors. Advances in the structural biology of a specific class of membrane receptors, the G protein-coupled receptors, have significantly increased our understanding of drug action and paved the way for developing improved therapeutic approaches. We have analyzed the effect of the flavonoid quercetin on the conformation, stability and function of the G protein-coupled receptor rhodopsin, and the G90V mutant associated with the retinal degenerative disease retinitis pigmentosa. By using a combination of experimental and computational methods, we suggest that quercetin can act as an allosteric modulator of opsin regenerated with 9-cis-retinal and more importantly, that this binding has a positive effect on the stability and conformational properties of the G90V mutant associated with retinitis pigmentosa. These results open new possibilities to use quercetin and other flavonoids, in combination with specific retinoids like 9-cis-retinal, for the treatment of retinal degeneration associated with retinitis pigmentosa. Moreover, the use of flavonoids as allosteric modulators may also be applicable to other members of the G protein-coupled receptors superfamily.