The GPCR signalling cascade is a key pathway responsible for the signal transduction of a multitude of physical and chemical stimuli, including light, odorants, neurotransmitters and hormones. Understanding the structural and functional properties of the GPCR cascade requires direct observation of signalling processes in high spatial and temporal resolution, with minimal perturbation to endogenous systems. Optical microscopy and spectroscopy techniques are uniquely suited to this purpose because they excel at multiple spatial and temporal scales and can be used in living objects. Here, we review recent developments in microscopy and spectroscopy technologies which enable new insights into GPCR signalling. We focus on advanced techniques with high spatial and temporal resolution, single-molecule methods, labelling strategies and approaches suitable for endogenous systems and large living objects. This review aims to assist researchers in choosing appropriate microscopy and spectroscopy approaches for a variety of applications in the study of cellular signalling. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.
- MeSH
- Humans MeSH
- Microscopy * methods MeSH
- Receptors, G-Protein-Coupled * chemistry metabolism MeSH
- Signal Transduction MeSH
- Spectrum Analysis * methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Adenosine Triphosphatases genetics metabolism MeSH
- Bacterial Proteins * metabolism MeSH
- Nucleotides metabolism MeSH
- SecA Proteins metabolism MeSH
- Escherichia coli Proteins * metabolism MeSH
- SEC Translocation Channels chemistry MeSH
- Protein Transport MeSH
- Publication type
- Journal Article MeSH
- MeSH
- Cell Differentiation MeSH
- Encephalitis, Tick-Borne * immunology virology MeSH
- Stem Cells virology immunology MeSH
- Langerhans Cells * immunology virology MeSH
- Humans MeSH
- Encephalitis Viruses, Tick-Borne * immunology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Letter MeSH
The influence of temperature on photosynthetic reactions was investigated by a combination of time-resolved bacteriochlorophyll fluorescence, steady-state and differential absorption spectroscopy, and polarographic respiration measurements in intact cells of purple non-sulphur bacterium Rhodospirillum rubrum. Using variable bacteriochlorophyll fluorescence, it was found that the electron-transport activity increased with the increasing temperature up to 41 °C. The fast and medium components of the fluorescence decay kinetics followed the ideal Arrhenius equation. The calculated activation energy for the fast component was Ea1 = 16 kJ mol-1, while that of the medium component was more than double, with Ea2 = 38 kJ mol-1. At temperatures between 41 and 59 °C, the electron transport was gradually, irreversibly inhibited. Interestingly, the primary charge separation remained fully competent from 20 to 59 °C as documented by both BChl fluorescence and differential absorption spectroscopy of the P870+ signal. At temperatures above 60 °C, the primary photochemistry became reversibly inhibited, which was manifested by an increase in minimal fluorescence, F0, whereas maximal fluorescence, FM, slowly declined. Finally, above 71 °C, the photosynthetic complexes began to disassemble as seen in the decline of all fluorometric parameters and the disappearance of the LH1 absorption band at 880 nm. The extended optimal temperature of photosynthetic reaction centre in a model species of Rhodospirillales adds on the evidence that the good thermostability of the photosynthetic reaction centres is present across all Alphaproteobacteria.
BACKGROUND: The availability of tick in vitro cell culture systems has facilitated many aspects of tick research, including proteomics. However, certain cell lines have shown a tissue-specific response to infection. Thus, a more thorough characterization of tick cell lines is necessary. Proteomic comparative studies of various tick cell lines will contribute to more efficient application of tick cell lines as model systems for investigation of host-vector-pathogen interactions. RESULTS: Three cell lines obtained from a hard tick, Ixodes ricinus, and two from I. scapularis were investigated. A cell mass spectrometry approach (MALDI-TOF MS) was applied, as well as classical proteomic workflows. Using PCA, tick cell line MS profiles were grouped into three clusters comprising IRE/CTVM19 and ISE18, IRE11 and IRE/CTVM20, and ISE6 cell lines. Two other approaches confirmed the results of PCA: in-solution digestion followed by nanoLC-ESI-Q-TOF MS/MS and 2D electrophoresis. The comparison of MS spectra of the cell lines and I. ricinus tick organs revealed 29 shared peaks. Of these, five were specific for ovaries, three each for gut and salivary glands, and one for Malpighian tubules. For the first time, characteristic peaks in MS profiles of tick cell lines were assigned to proteins identified in acidic extracts of corresponding cell lines. CONCLUSIONS: Several organ-specific MS signals were revealed in the profiles of tick cell lines.
- MeSH
- Electrophoresis, Gel, Two-Dimensional MeSH
- Cell Line * cytology metabolism MeSH
- Insect Proteins metabolism MeSH
- Ixodes cytology MeSH
- Proteomics MeSH
- Salivary Glands cytology MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * MeSH
- Tandem Mass Spectrometry MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.
- MeSH
- Bacterial Proteins chemistry genetics metabolism MeSH
- Escherichia coli genetics metabolism MeSH
- Gene Expression MeSH
- Genetic Vectors chemistry metabolism MeSH
- Hydrocarbons, Halogenated chemistry metabolism MeSH
- Hydrolases chemistry genetics metabolism MeSH
- Protein Interaction Domains and Motifs MeSH
- Cloning, Molecular MeSH
- Protein Conformation, alpha-Helical MeSH
- Protein Conformation, beta-Strand MeSH
- Crystallography, X-Ray MeSH
- Cold Temperature MeSH
- Psychrobacter chemistry enzymology MeSH
- Recombinant Fusion Proteins chemistry genetics metabolism MeSH
- Amino Acid Sequence MeSH
- Molecular Docking Simulation MeSH
- Structural Homology, Protein MeSH
- Substrate Specificity MeSH
- Thermodynamics MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Publication type
- Journal Article MeSH
Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide (Allen et al., 2016). Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogendeuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP-induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.
- MeSH
- Adenosine Triphosphate chemistry metabolism MeSH
- Adenosine Triphosphatases chemistry metabolism MeSH
- Escherichia coli metabolism MeSH
- Membrane Transport Proteins chemistry metabolism MeSH
- Models, Molecular MeSH
- Protein Precursors metabolism MeSH
- SecA Proteins chemistry metabolism MeSH
- Escherichia coli Proteins chemistry metabolism MeSH
- Protein Folding * MeSH
- SEC Translocation Channels chemistry metabolism MeSH
- Protein Transport MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
