Signal transduction in light-oxygen-voltage receptors lacking the adduct- forming cysteine residue

Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct- forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications

Abstract High resolution electron spin resonance microscopy

NMR microscopy is routinely employed in fields of science such as biology, botany, and materials science to observe magnetic parameters and transport phenomena in small scale structures. Despite extensive efforts, the resolution of this method is limited (>10lm for short acquisition times), and thus cannot answer many key questions in these fields. We show, through theoretical prediction and initial experiments, that ESR microscopy, although much less developed, can improve upon the resolution limits of NMR, and successfully undertake the 1lm resolution challenge. Our theoretical predictions demonstrate that existing ESR technology, along with advanced imaging probe design (resonator and gradient coils), using solutions of narrow linewidth radicals (the trityl family), should yield 64 64 pixels 2D images (with z slice selection) with a resolution of 1 1 10lmat 60 GHz in less than 1 h of acquisition. Our initial imaging results, conducted by CW ESR at X-band, support these theoretical predictions and already improve upon the previously reported state-of-the-art for 2D ESR image resolution achieving 10 10lm, in just several minutes of acquisition time. We analyze how future progress, which includes improved resonators, increased frequency of measurement, and advanced pulsed techniques, should achieve the goal of micron resolution

Improved Sensitivity for Long-Distance Measurements in Biomolecules: Five-Pulse Double Electron–Electron Resonance

We describe significantly improved long-distance measurements in biomolecules by use of the new multipulse double electron–electron spin resonance (DEER) illustrated with the example of a five-pulse DEER sequence. In this sequence, an extra pulse at the pump frequency is used compared with standard four-pulse DEER. The position of the extra pulse is fixed relative to the three pulses of the detection sequence. This significantly reduces the effect of nuclear spin-diffusion on the electron-spin phase relaxation, thereby enabling longer dipolar evolution times that are required to measure longer distances. Using spin-labeled T4 lysozyme at a concentration less than 50 μM, as an example, we show that the evolution time increases by a factor of 1.8 in protonated solution and 1.4 in deuterated solution to 8 and 12 μs, respectively, with the potential to increase them further. This enables a significant increase in the measurable distances, improved distance resolution, or both

Open and Closed Form of Maltose Binding Protein in Its Native and Molten Globule State As Studied by Electron Paramagnetic Resonance Spectroscopy

An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP. DEER data nicely corroborated the previously available X-ray data. Even in its MG state, MBP is known to still bind its ligand maltose. We therefore hypothesized that there must be a defined structure around the binding pocket of MBP already in the absence of tertiary structure. Here we have investigated the functional and structural difference between native and MG state in the open and closed form with a new set of MBP mutants. In these, the spin-label positions were placed near the active site. Binding of its ligands leads to a conformational change from open to closed state, where the two domains are more closely together. The complete set of MBP mutants was analyzed at pH 3.2 (MG) and pH 7.4 (native state) using double-quantum coherence EPR. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structures of MBP in open and closed form and were found to be in excellent agreement. Measurements show a defined structure around the binding pocket of MBP in MG, which explains maltose binding. A new and important finding is that in both states ligand-free MBP can be found in open and closed form, while ligand-bound MBP appears only in closed form because of maltose binding

Conformational motion of the ABC transporter MsbA induced by ATP hydrolysis.

We measured the amplitude of conformational motion in the ATP-binding cassette (ABC) transporter MsbA upon lipopolysaccharide (LPS) binding and following ATP turnover by pulse double electron-electron resonance and fluorescence homotransfer. The distance constraints from both methods reveal large-scale movement of opposite signs in the periplasmic and cytoplasmic part of the transporter upon ATP hydrolysis. LPS induces distinct structural changes that are inhibited by trapping of the transporter in an ATP post-hydrolysis intermediate. The formation of this intermediate involves a 33-A distance change between the two ABCs, which is consistent with a dimerization-dissociation cycle during transport that leads to their substantial separation in the absence of nucleotides. Our results suggest that ATP-powered transport entails LPS sequestering into the open cytoplasmic chamber prior to its translocation by alternating access of the chamber, made possible by 10-20-A conformational changes

HIV-1 Vpu protein forms stable oligomers in aqueous solution via its transmembrane domain self-association

Abstract We report our findings on the assembly of the HIV-1 protein Vpu into soluble oligomers. Vpu is a key HIV-1 protein. It has been considered exclusively a single-pass membrane protein. Previous observations show that this protein forms stable oligomers in aqueous solution, but details about these oligomers still remain obscure. This is an interesting and rather unique observation, as the number of proteins transitioning between soluble and membrane embedded states is limited. In this study we made use of protein engineering, size exclusion chromatography, cryoEM and electron paramagnetic resonance (EPR) spectroscopy to better elucidate the nature of the soluble oligomers. We found that Vpu oligomerizes via its N-terminal transmembrane domain (TM). CryoEM suggests that the oligomeric state most likely is a hexamer/heptamer equilibrium. Both cryoEM and EPR suggest that, within the oligomer, the distal C-terminal region of Vpu is highly flexible. Our observations are consistent with both the concept of specific interactions among TM helices or the core of the oligomers being stabilized by hydrophobic forces. While this study does not resolve all of the questions about Vpu oligomers or their functional role in HIV-1 it provides new fundamental information about the size and nature of the oligomeric interactions

Conformational response of influenza A M2 transmembrane domain to amantadine drug binding at low pH (pH 5.5)

The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21-49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21-49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21-49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21-49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L’s ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5-8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD21-49 through increased motional ordering. In contrast to wild-type M2TMD21-49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation found in certain drug-resistant influenza strains. Thus, the inhibited M2TMD21-49 channel is a stable tetramer with a closed C-terminal exit pore. This work is aimed at contributing to the development of structure-based anti-influenza pharmaceuticals