Cyclic nucleotide-regulated ion channels are present in bacteria, plants, vertebrates, and humans. In higher organisms, they are closely involved in signaling networks of vision and olfaction. Binding of cAMP or cGMP favors the activation of these ion channels. Despite a wealth of structural and studies, there is a lack of structural data describing the gating process in a full-length cyclic nucleotideregulated channel. We used high-resolution atomic force microscopy (AFM) to directly observe the conformational change of the membrane embedded bacterial cyclic nucleotide-regulated channel MlotiK1. In the nucleotide-bound conformation, the cytoplasmic cyclic nucleotide-binding (CNB) domains of MlotiK1 are disposed in a fourfold symmetric arrangement forming a pore-like vestibule. Upon nucleotide-unbinding, the four CNB domains undergo a large rearrangement, stand up by ∼1.7 nm, and adopt a structurally variable grouped conformation that closes the cytoplasmic vestibule. This fully reversible conformational change provides insight into how CNB domains rearrange when regulating the potassium channel.conformational changes | cyclic nucleotide gating | membrane protein | MloK1 | single-molecule imaging P otassium channels are tetrameric membrane proteins that facilitate the permeation of potassium ions through the membrane with high specificity and high-throughput rates. These channels are central to the electrical activity of cells in humans and are, therefore, of fundamental importance for the function of nervous and muscular systems. The major mode of functional regulation in potassium channels is gating, a conformational change that occurs on the intracellular regions of the ion pore domain and involves an iris-like movement of the C-terminal transmembrane helices and a widening of the intracellular pore. Gating in potassium channels is induced by a variety of stimuli, including membrane voltage, intracellular calcium concentration, and cyclic nucleotide levels (1). These stimuli are sensed by a separate domain from the ion pore domain, inducing a conformational change that is then propagated to the gate of the channel.The MlotiK1 potassium channel, from the bacterium Mesorhizobium loti, belongs to the family of channels that is regulated by cyclic nucleotides and includes eukaryotic cyclic nucleotide-gated (CNG) and hyperpolarization activated cyclic nucleotide-gated (HCN) channels (2, 3). These channels have C-terminal cytoplasmic cyclic nucleotide-binding (CNB) domains and upon binding of cAMP or cGMP, these domains undergo a conformational change that favors the opening of the gate of the channel. The major difference between the MlotiK1 channel and the CNG or HCN channels is the linker that connects the gate to the CNB domains. This helical linker (C linker) is roughly 80 residues long in CNG and HCN channels and only ∼20 residues long in the MlotiK1 channel.The MlotiK1 channel has been the focus of structural and functional studies with the aim of understanding channel regulation by cyclic nucleotides. X-ray...
BackgroundArabidopsis thaliana transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL.ResultsThe TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein.ConclusionsThe Arabidopsis thaliana transthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.
Temporal and spatial variations and environmental factors influencing the structure of tidepool fish assemblages were quantitatively investigated at Iparana beach, northeast Brazilian coast. The majority of the tidepool fishes sampled were suprabenthic juvenile individuals of great mobility. We recorded during monthly diurnal underwater visual censuses a total of 4,750 fish from 26 species, represented mainly by partial residents from the families Scaridae, Haemulidae, Gerreidae and Pomacentridae. The number of species and individuals showed significant variability among tidepools as a response to variations in their volume and type of substrate cover. The greatest species abundance and richness associated with rocks covered with algae suggested that substrate complexity is one of the main factors defining the spatial structure of the tidepool ichthyofauna. Temporal variability in species abundance and richness was associated with changes in salinity levels due to seasonal rainfalls. A higher number of juvenile fishes from December to May in our samples corroborate the hypothesis that the tidepools act as nursery sites. Therefore, the species associations found in this study and their relation to seasonal and spatial discontinuities may be partly explained according to their habitat requirements and reproductive cycles.
This work combined water equilibration fundamentals of vapor diffusion crystallization techniques with protein solubility data in order to obtain the variation of protein supersaturation throughout the protein crystallization assays. Once the supersaturation build up profiles (SBUPs) are known, the wide screening space of crystallization conditions is reduced to the key variable for crystal formation and growth, which is supersaturation and its variation with time. Our previous water equilibration model was expanded to include the case of drop evaporation at constant contact area during the hanging drop method. Crystallization experiments of lysozyme were performed under different experimental conditions and the results were interpreted according to the respective SBPUs. In particular, the number and size of the crystals were evaluated at the moment of the SBUP that corresponded to their formation. Following this methodology, two nucleation behaviors were identified depending on the supersaturation levels at which crystal formation occurs. These behaviors, which are believed to be closely linked with the diffracting properties of the crystals, are dictated not only by classic thermodynamic and kinetic factors affecting crystallization and water equilibration, but also by phenomena related to the drop preparation procedures.
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