ABSTRACT
The voltage-dependent anion channel (VDAC) is the most abundant protein of the outer mitochondrial membrane and constitutes the major pathway for the transport of ADP, ATP, and other metabolites. In this multidisciplinary study we combined solid-state NMR, electrophysiology, and molecular dynamics simulations, to study the structure of the human VDAC isoform 2 in a lipid bilayer environment. We find that the structure of hVDAC2 is similar to the structure of hVDAC1, in line with recent investigations on zfVDAC2. However, hVDAC2 appears to exhibit an increased conformational heterogeneity compared to hVDAC1 which is reflected in broader solid-state NMR spectra and less defined electrophysiological profiles.
Subject(s)
Electrophysiological Phenomena/physiology , Nuclear Magnetic Resonance, Biomolecular/methods , Voltage-Dependent Anion Channel 1/ultrastructure , Voltage-Dependent Anion Channel 2/chemistry , Voltage-Dependent Anion Channel 2/ultrastructure , Amino Acid Sequence , Humans , Lipid Bilayers/chemistry , Mitochondria/metabolism , Molecular Conformation , Molecular Dynamics Simulation , Molecular Sequence Data , Protein Isoforms/chemistry , Sequence AlignmentABSTRACT
Membrane proteins play key roles in biology. Determination of their structure in a membrane environment, however, is highly challenging. To address this challenge, we developed an approach that couples hydrogen/deuterium exchange of membrane proteins to rapid unfolding and detection by solution-state NMR spectroscopy. We show that the method allows analysis of the solvent protection of single residues in liposome-embedded proteins such as the 349-residue Tom40, the major protein translocation pore in the outer mitochondrial membrane, which has resisted structural analysis for many years.
Subject(s)
Fungal Proteins/chemistry , Liposomes/chemistry , Membrane Proteins/chemistry , Mitochondrial Proteins/chemistry , Neurospora crassa/chemistry , Protein Unfolding , Deuterium Exchange Measurement , Nuclear Magnetic Resonance, BiomolecularABSTRACT
The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.
