Influenza hemagglutinin is the fusion protein that mediates fusion of the viral and host membranes through a large conformational change upon acidification in the developing endosome. The ...“spring-loaded” model has long been used to describe the mechanism of hemagglutinin and other type 1 viral glycoproteins. This model postulates a metastable conformation of the HA2 subunit, caged from adopting a lower-free energy conformation by the HA1 subunit. Here, using a combination of biochemical and spectroscopic methods, we study a truncated construct of HA2 (HA2*, lacking the transmembrane domain) recombinantly expressed in Escherichia coli as a model for HA2 without the influence of HA1. Our data show that HA2* folds into a conformation like that of HA2 in full length HA and forms trimers. Upon acidification, HA2* undergoes a conformational change that is consistent with the change from pre- to postfusion HA2 in HA. This conformational change is fast and occurs on a time scale that is not consistent with aggregation. These results suggest that the prefusion conformation of HA2 is stable and the change to the postfusion conformation is due to protonation of HA2 itself and not merely uncaging by HA1.
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FeFe-hydrogenases catalyze the reversible production of hydrogen gas from protons and electrons, but the mechanism of catalysis is still the subject of debate. In this report, we describe a ...pre-steady-state photoreduction methodology for correlating putative intermediates to the reactivity of an FeFe-hydrogenase from Thermotoga maritima. In this method, MV+ is rapidly formed photochemically by pulsed laser excitation and the intermolecular ET and active site dynamics are followed by nanosecond time-resolved visible and infrared spectroscopy, respectively. The results kinetically validate the Hox, Hred, and Hsred intermediates, strongly supporting a vast body of literature on their involvement in proton reduction.
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Helices 2 and 3 of Engrailed homeodomain (EnHD) form a helix-turn-helix (HTH) motif. This common motif is believed not to fold independently, which is the characteristic feature of a motif rather ...than a domain. But we found that the EnHD HTH motif is monomeric and folded in solution, having essentially the same structure as in full-length protein. It had a sigmoidal thermal denaturation transition. Both native backbone and local tertiary interactions were formed concurrently at 4 x 10⁵ s⁻¹ at 25°C, monitored by IR and fluorescence T-jump kinetics, respectively, the same rate constant as for the fast phase in the folding of EnHD. The HTH motif, thus, is an ultrafast-folding, natural protein domain. Its independent stability and appropriate folding kinetics account for the stepwise folding of EnHD, satisfy fully the criteria for an on-pathway intermediate, and explain the changes in mechanism of folding across the homeodomain family. Experiments on mutated and engineered fragments of the parent protein with different probes allowed the assignment of the observed kinetic phases to specific events to show that EnHD is not an example of one-state downhill folding.
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Las islas Desventuradas (San Félix y San Ambrosio) y del Archipiélago de Juan Fernández (Robinson Crusoe y Alejandro Selkirk) son islas oceánicas de origen volcánico ubicadas en el Pacífico Sudeste, ...frente a Chile. Sobre la base de material recolectado desde 1997 hasta la fecha, revisiones de colecciones de las expediciones del R/V Anton Bruun y actualizaciones taxonómicas, la fauna de peces litorales del Archipélago de Juan Fernández y las islas Desventuradas tienen un total de 52 especies, con 41 y 43 especies, respectivamente. Los peces costeros endémicos de Juan Fernández son solamente cinco (12,2%) y tres son las especies registradas aquí como nuevas para las islas (Gymnothorax cf. obesus, Gnathophis sp., Suezichthys sp.). Las Desventuradas tienen solamente dos especies endémicas (4,6%) y cuatro son los nuevos registros (Scorpaenodes englerti, Maxillicosta reticulata, Suezichthys sp., Aseraggodes bahamondei). El reducido número de especies endémicas para Juan Fernández comparado con trabajos previos (15 especies) se debe a que muchas resultaron ser endémicas de ambos grupos de islas (22 especies = 42,3% total peces litorales), apoyando la propuesta que ambos grupos de islas debieran formar una sola unidad biogeográfica. La extensión oriental de la región Indo Pacífico Oeste para incluir la cordillera de Nazca, las islas Desventuradas y Juan Fernández encuentra apoyo en este trabajo. La afinidad zoogeográfica entre las Desventuradas y las islas del Pacífico Oeste (Isla de Pascua hasta Australia) incluye 16 especies, agregando cinco nuevos registros a listas anteriores. Filogenias de tres géneros señalan un centro de origen en el Pacífico Oeste, con uno o más eventos de dispersión hacia las islas Desventuradas y Juan Fernández. Este patrón de dispersión puede repetirse en otros siete géneros de pocas especies y relativamente restringidas en distribución. Solo una filogenia (Odontesthes) propone un origen continental con una dispersión hacia el Oeste, sin embargo otros tres géneros parecen tener este mismo patrón de dispersión.
The Desventuradas Islands (San Félix and San Ambrosio) and Juan Fernández Archipelago (Robinson Crusoe and Alejandro Selkirk) are oceanic islands of volcanic origin located in the southeastern Pacific off Chile. Based on new material collected since 1997, revision of the R/V Anton Bruun expeditions collections, and a taxonomic update, the coastal fish fauna of Juan Fernández Archipelago and Desventuradas Islands found a total of 52 species, with 41 and 43 species, respectively. The number of coastal fish species recorded herein as endemic for Juan Fernández is only five (12,2%) and three species are new records (Gymnothorax cf. obesus, Gnathophis sp., Suezichthys sp). The Desventuradas Islands have only two endemic species (4,6%) and four species are new records (Scorpaenodes englerti, Maxillicosta reticulata, Suezichthys sp., Aseraggodes bahamondei). The reduced number of endemic species for the Juan Fernández Islands as compared with previous accounts (15 species) is the result that more species were found to be endemic to both islands groups (22 species = 42,3% all coastal fishes), suggesting these two island groups should be considered a single biogeographic unit. The eastern extension of the Indo West Pacific Region to include the Nazca Ridge, Desventuradas and Juan Fernández Islands is supported herein. The zoogeographical affinities between Desventuradas and western Pacific Islands (Easter Island to Australia), includes 16 species, adding five new species to previous lists. Relationships of three genera indicate a Western Pacific origin with one or more dispersal events to the Desventuradas and Juan Fernández Islands. Another seven genera, with relatively few species and restricted distribution, may have this same pattern. Only one phylogenetic study indicates a continental origin with a westward dispersal (Odontesthes), however another three genera may have this same pattern of dispersal.
The M2 proton transport channel of the influenza virus A is an important model system because it conducts protons with high selectivity and unidirectionally when activated at low pH, despite the ...relative simplicity of its structure. Although it has been studied extensively, the molecular details of the pH-dependent gating and proton conductance mechanisms are incompletely understood. We report direct observation of the M2 proton channel activation process using a laser-induced pH jump coupled with tryptophan fluorescence as a probe. Biphasic kinetics is observed, with the fast phase corresponding to the His37 protonation, and the slow phase associated with the subsequent conformation change. Unusually fast His37 protonation was observed (2.0 × 1010 M–1 s–1), implying the existence of proton collecting antennae for expedited proton transport. The conformation change (4 × 103 s–1) was about 2 orders of magnitude slower than protonation at endosomal pH, suggesting that a transporter model is likely not feasible.
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Equilibrium Fourier transform infrared (FTIR) and temperature-jump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin ...headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L, F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L, F51L. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WT (3.23 µs at 60.2°C) and double phenylalanine mutant (3.01 µs at 49.9°C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 µts at 49.9µC, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant, however, unfolds faster with an unfolding time of 3.01 µs compared with 6.97 µs for the WT at 49.9°C.
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Hydrogenases are metalloenzymes that catalyze the reversible oxidation of H2. The FeFe hydrogenases are generally biased toward proton reduction and have high activities. Several different catalytic ...mechanisms have been proposed for the FeFe enzymes based on the identification of intermediate states in equilibrium and steady state experiments. Here, we examine the kinetic competency of these intermediate states in the FeFe hydrogenase from Chlamydomonas reinhardtii (CrHydA1), using a laser-induced potential jump and time-resolved IR (TRIR) spectroscopy. A CdSe/CdS dot-in-rod (DIR) nanocrystalline semiconductor is employed as the photosensitizer and a redox mediator efficiently transfers electrons to the enzyme. A pulsed laser induces a potential jump, and TRIR spectroscopy is used to follow the population flux through each intermediate state. The results clearly establish the kinetic competency of all intermediate populations examined: Hox, Hred, HredH+, HsredH+, and Hhyd. Additionally, a new short-lived intermediate species with a CO peak at 1896 cm–1 was identified. These results establish a kinetics framework for understanding the catalytic mechanism of FeFe hydrogenases.
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