Search

Summary The spontaneous insertion of helical transmembrane (TM) polypeptides into lipid bilayers is driven by three sequential equilibria: solution-to-membrane interface (MI) partition, unstructured-to-helical folding, and MI-to-TM helix insertion. A bottleneck for understanding these three steps is the lack of experimental approaches to perturb membrane-bound hydrophobic polypeptides out of equilibrium rapidly and reversibly. Here, we report on a 24-residues-long hydrophobic α-helical polypeptide, covalently coupled to an azobenzene photoswitch (KCALP-azo), which displays a light-controllable TM/MI equilibrium in hydrated lipid bilayers. FTIR spectroscopy reveals that trans KCALP-azo folds as a TM α-helix (TM topology). After trans-to-cis photoisomerization of the azobenzene moiety with UV light (reversed with blue light), the helical structure of KCALP-azo is maintained, but its helix tilt increased from 32 ± 5° to 79 ± 8°, indication of a reversible TM-to-MI transition. Further analysis indicates that this transition is incomplete, with cis KCALP-azo existing in a ∼90% TM and ∼10% MI mixture., Graphical abstract, Highlights • We present an α-helical transmembrane peptide modified with a molecular photoswitch • The peptide exhibits reversible photocontrol of its membrane topology • A fraction moves to the membrane interface with UV and inserts back with blue light • This system will be useful to address the molecular mechanism for membrane insertion, Photoabsorption; Biomolecules; Membranes

Translocation of protease into mesoporous MIL-101-NH2 results in enhanced catalytic activity, excellent recyclability and tolerance to competing enzymes., Enzymatic catalysis is of great importance to the chemical industry. However, we are still scratching the surface of the potential of biocatalysis due to the limited operating range of enzymes in harsh environments or their low recyclability. The role of Metal–Organic Frameworks (MOFs) as active supports to help overcome these limitations, mainly by immobilization and stabilization of enzymes, is rapidly expanding. Here we make use of mild heating and a non-polar medium during incubation to induce the translocation of a small enzyme like protease in the mesoporous MOF MIL-101(Al)-NH2. Our proteolytic tests demonstrate that protease@MIL-101(Al)-NH2 displays higher activity than the free enzyme under all the conditions explored and, more importantly, its usability can be extended to extreme conditions of pH and high temperatures. MOF immobilization is also effective in providing the biocomposite with long-term stability, recyclability and excellent compatibility with competing enzymes. This simple, one-step infiltration strategy might accelerate the discovery of new MOF-enzyme biocatalysts that meet the requirements for biotechnological applications.

Channelrhodopsins (ChRs) are light-gated cation channels. In spite of their wide use to activate neurons with light, the photocurrents of ChRs rapidly decay in intensity under both continuous illumination and fast trains of light pulses, broadly referred to as desensitization. This undesirable phenomenon has been explained by two interconnected photocycles, each of them containing a nonconductive dark state (D1 and D2) and a conductive state (O1 and O2). While the D1 and O1 states correspond to the dark-state and P3520 intermediate of the primary all-trans photocycle of ChR2, the molecular identity of D2 and O2 remains unclear. We show that P4480, the last intermediate of the all-trans photocycle, is photoactive. Its photocycle, characterized by time-resolved UV/vis spectroscopy, contains a red-shifted intermediate, I3530. Our results indicate that the D2 and O2 states correspond to the P4480 and I3530 intermediates, connecting desensitization of ChR2 with the photochemical properties of the P4480 intermediate.

Zeolites NaY and ZSM-5 were used as hosts for styrene polymerization after ion-exchange with europium ions. The parent and hybrid, polystyrene coated Eu-NaY (Eu-NaY/PS) and Eu-ZSM-5 (Eu-ZSM-5/PS) zeolites were investigated by using thermal analysis, SEM, PXRD, FT-IR, DR-UV/Vis, steady state and time-resolved photoluminescence spectroscopy. FT-IR spectra evidenced for the interaction between the zeolitic hosts and polystyrene while the PXRD spectra supported for the presence of the polymer inside the channels/pores of Eu-NaY/PS and Eu-ZSM-5/PS materials. The optical properties of Eu-NaY/PS and Eu-ZSM-5/PS were significantly changed relative to those of the parent zeolites, giving further evidence for the presence of polymer inside zeolites. An interesting case is presented by NaY zeolite: following styrene polymerization, the polymer interacted selectively with one of the two main species co-existing inside zeolite while for ZSM-5 a similar effect was not observed.

En este trabajo, el objetivo principal ha sido el estudio del mecanismo del co-transporte de la permeasa de melibiosa (MelB) de Escherichia coli. Este proteina de membrana realiza co-transporte activo de varios oligosacáridos utilizando la fuerza electroquímica de cationes. Una característica remarcable de este transportador es el hecho que permite utilizar tres diferentes cationes, el protón, el sodio y el litio. Los sustratos con mayor afinidad a la proteína son el sodio y la melibiosa. Para cumplir nuestro objetivo principal se llevó a cabo el estudio de los dominios C-terminales de la proteína reconstituida como proteoliposomas. El bucle existente entre las hélices 10 y 11 así como la hélice XI del transportador se revelaron como dominios fundamentales para el funcionamiento del transporte activo. Estudios previos indican que la substitución de diversos amino ácidos por una cisteína impide el transporte. Con la técnica de mutagénesis dirigida se obtuvieron substituciones de amino ácidos en la proteína MelB, generando así múltiples mutantes con diferentes propiedades de unión, de translocación y de liberación de los sustratos. La solubilizacion de los mutantes generados se realizó con los detergentes LAPAO y DDM. Después de varios pasos de purificación, los mutantes de la MelB se reconstituyeron en liposomas de Escherichia coli para simular así el hábitat natural del transportador. Con técnicas espectroscópicas (infrarrojo y fluorescencia) se analizaron la unión de diferentes mutantes de MelB a los sustratos. Este estudio puso de manifiesto el papel crucial del amino ácido Lys-377 en el transportador MelB. Substituciones de dicho amino ácido por cisteína, valina, histidina, arginina y ácido aspártico mostraron como el transportador perdía completamente la unión a sodio y pasaba a acoplar el azúcar sólo en presencia del protón pero de manera disminuida. La carga en posición 377 de la MelB destaca por mantener el acoplamiento a los sustratos. Otra característica importante de la MelB es el volumen de cadena lateral que influye en la unión a sodio y melibiosa. La búsqueda de revertantes se realizó a través de un fenotipo diferente de la cepa, haciéndola crecer en placas de agar suplementadas de melibiosa. Los mutantes con un defecto de transporte, como es el caso de los mutantes de Lys-377, eran reconocidos al formar éstos colonias blancas, a diferencia de los revertantes, que generaban colonias rojas, indicando este color un pH dentro de la célula distinto en ambos casos. El cambio rojo en los revertantes es debido a la hidrólisis de la melibiosa en dos monosacáridos, glucosa y galactosa. La hidrólisis disminuye el pH que es detectado por el indicador suplementado en la placa. La secuenciación de estas subcolonias rojas reveló substituciones de los amino ácidos Ile-22 por serina y Leu-236 por fenilalanina. La mutación I22S ocurre en todos mutantes singulares de Lys-377, pero el desplazamiento de Leu-236 por fenialalanina sólo se detectó en el mutante K377R. Ninguno de los revertantes de K377/I22S recuperó la unión al azúcar en proteoliposomas y únicamente K377C/I22S fue capaz de unir el sodio pero con una afinidad menor. Los espectros de absorbancia de los revertantes indicaron que existe una cierta correlación entre cambios conformacionales de la MelB y la ausencia de señales en espectros de diferencia provocada por sodio. Como controles se usaron los mutantes singulares I22S y I22A que como revelan los espectros de diferencia, mantienen la capacidad de unión a el sodio pero no al azúcar. La isoleucina en posición 22 podría formar parte del sitio de unión de la melibiosa en MelB. Solubilizando el revertante K377R/L236F con LAPAO, detergente común para la purificación de MelB, demostró que dicho mutante tiene una afinidad disminuida a sus sustratos, tal y como demuestran los espectros de diferencia y fluorescencia. Sin embargo, las vesículas del mutante K377R/L236F tienen la capacidad de unir un análogo fluorescente de azúcar similar al C-less, la wild-type permeasa sin cisteínas. La solubilización del mutante con DDM unía los sustratos con mucha más afinidad que el mismo mutante solubilizado con LAPAO y mantienía la estructura similar a C-less. La prueba con K377C/L236F, un doble mutante generado por PCR, no reveló interacciones con los sustratos. Este resultado indica la importancia de la carga positiva conservada de la arginina en combinación con cambios estructurales para mantener la capacidad de unión de los sustratos. En la segunda parte se examinaron mutaciones con cisteínas de varios amino acidos cargados colocados en el bucle entre las hélices transmembranales X y XI. Estudios previos del transporte activo indican que tres amino ácidos perdían la capacidad de acumular melibiosa contra el gradiente. Los residuos Asp-351, Asp-354 y Arg-363 estudiados a través de IRdiff y fluorescencia, demuestraron la persistencia de unión a sodio aunque reducida. Los tres mutantes de MelB unían el azúcar también pero de una forma drásticamente reducida. Sólo en un revertante se descubrió un desplazamiento también en Ile-22 por una serina como en el caso de Lys-377. El revertante D354C/I22S demostró un incremento de la unión a sodio, pero una pérdida total de la unión a melibiosa. En el parte final de la tesis, se generaron ocho mutantes. Cada mutante con una mutación en un amino ácido diferente colocados todos ellos en la hélice XI de la MelB. Q372C, G379C, F385C, L391C y G395C mantuvieron la unión de sodio de manera equivalente a C-less. A383C y Y396C mostraron una cierta reducción de la señal mediada por sodio. El mutante G379V fue el único mutante que perdió totalmente la interacción con el catión. Los espectros de IRdiff inducido por melibiosa demostraron en A383C, L391C y Y396C una afinidad muy baja al azúcar. G379V tampoco fue capaz de unir la melibiosa., This doctoral thesis was dedicated to the study of the secondary transporter, the melibiose permase (MelB) from Escherichia coli with biochemical and biophysical methodologies. The main objective was to obtain insights of the symport mechanism of MelB. This prokaryotic transporter uses the downhill translocation of a cation to transport the disaccharide melibiose against its concentration gradient into the cell. Although MelB possess the highest affinity for Na+ cations, the transporter can couple the transport process also to the smaller Li+ and H+ ions. Apart from melibiose, MelB transports a variety of α- and β-galactosides making it versatile carrier. The transporter comprise 70% of hydrophobic amino acids and is organized in 12 transmembrane spanning helices connected by hydrophilic loops. As preliminary studies already reveal, the C-terminal domains of MelB has been proven to play a crucial role in the active transport mechanism. The focus in this doctoral thesis lies especially on the helices X and XI as well as the interconnecting cytoplasmic loop 10-11. Site-directed mutagenesis delivered valuable information about important amino acids which might participate in the active transport by forming part of the binding sites or taking part in the translocation and release of the substrates. During our study, numerous MelB mutants have been extracted from the membrane by using the detergents LAPAO and β-DDM. Subsequently, the solubilized transporter was reconstituted into liposomes composed of lipids from E. coli mimicking MelB’s original habitat. As the only charged residue residing in the transmembrane segment XI, Lys-377 was replaced by cysteine, valine, arginine, histidine and aspartic acid. None of these MelB mutants interacts with sodium concluded from infrared difference spectra (IRdiff) induced by the cation Na+. The melibiose binding is only remotely detectable in K377R, K377H and K377C in the presence of the proton. K377V and K377D lost the interaction with the sugar molecule. The charge in the particular position in MelB is important but not sufficient to conserve the substrate binding. The amino acid substitutions of Lys-377 appear as white colonies on a MacConkey agar plate. This color indicates the absence of hydrolysis of the melibiose into the monosaccharides, glucose and galactose. A metabolisation of the sugar is indicated by a red colony. In these colored bacteria, the sugar hydrolysis causes an acidification of the medium indicated by the MacConkey agar which stains the bacterial colonies. The screening of potential revertants, second site mutations which allow sugar influx, demonstrate the appearance of a unique second site mutation I22S which turn the former white colonies of the Lys-377 mutants into a red phenotype. Another red-colored phenotype appeared only for the mutant K377R, in which Leu-236 is replaced by a phenylalanine. All K377/I22S revertants are not able to restore the melibiose binding in proteoliposomes and only the revertant K377C/I22S recovered a partial sodium binding. The absorbance spectra of the single Lys-mutants as well as the corresponding revertants indicate a correlation between the absence of sodium binding and conformational changes of the MelB transporter. These structural changes are identical to previously examined MelB mutants D55C, D59C and D124C which also demonstrated a detrimental impact on the Na+ binding. The single mutants I22S and I22A exhibit a clear response of the structure in the presence of Na+. The interaction with melibiose on the contrary is lost in these MelB mutants. This outlines Ile-22 as a potential amino acid participating in the binding process of the melibiose molecule in MelB. The second revertant K377R/L236F demonstrates only minor interaction with the Na+ as well with the melibiose in proteoliposomes. Measurements using membrane vesicles of this mutant demonstrate however a large Förster energy transfer (FRET) signal mediated by the fluorescent sugar analog, D2G. By extracting the mutant K377R/L236F with β-DDM instead of LAPAO, the solubilised and then reconstituted transporter exhibits a clear improvement considering the conservation of the MelB structure. IRdiff spectroscopy results concluded that the K377R/L236F mutant binds melibiose and Na+, although the cation-induced difference spectra differs from the C-less reference. The PCR-generated double mutant K377C/L236F failed to demonstrate similar interactions with the substrates in proteoliposomes as well as in membrane vesicles. The positive charge in position 377 is essential for the MelB transporter, but the sole charge is not sufficient for the substrate binding. The charge requires also structural arrangement to interact with the sugar and the co-ion. In the second part of the PhD thesis, cysteine mutants replacing charged residue in the cytoplasmic loop 10-11 were characterized for their ability of substrate binding. The transport-defective mutants D351C, D354C and R363C couple sodium with reduced affinity indicated by their low intense IRdiff spectra. The melibiose binding is even further reduced in these MelB mutants. Appearing as white colonies on MacConkey agar plate, the screening of potential revertants only revealed a second site mutation for D354C. Interestingly, Ile-22 was substituted for a serine like in the Lys-377 mutants. Congruent results with the K377/I22S mutants indicate for D354C/I22S the loss of sugar binding. However, the sodium triggers a difference spectrum similar to C-less and much more intense than in the D354C single mutant. This indicates a tight interaction of the cation with the MelB mutant D354C/I22S. In the third part of this thesis, eight mutants were generated by replacing crucial residues in the C-terminal helix XI of MelB with cysteine. The mutants Q372C, G379C, F385C, L391C and G395C show an almost normal interaction with Na+. A383C and Y396C on the other side display a Na+-induced IRdiff with low intensity indicating their impaired cation binding. Considering the interaction with melibiose, all helix XI mutants bind the sugar in the presence of either the proton or Na+. The MelB mutants A383C, L391C and Y396C exhibit a melibiose-induced difference spectrum with very low intensity indicating their importance for the binding process. Tested in vesicles and proteoliposomes, G379V is the only characterised mutant which lacks sodium and sugar binding making this mutant a potential candidate for crystallography trials to obtain the structure of the empty MelB transporter.