Franz Bartl

Monitoring Light-induced Structural Changes of Channelrhodopsin-2 by UV-visible and Fourier Transform Infrared Spectroscopy

Channelrhodopsin-2 (ChR2) is a microbial type rhodopsin and a light-gated cation channel that controls phototaxis in Chlamydomonas. We expressed ChR2 in COS-cells, purified it, and subsequently investigated this unusual photoreceptor by flash photolysis and UV-visible and Fourier transform infrared difference spectroscopy. Several transient photoproducts of the wild type ChR2 were identified, and their kinetics and molecular properties were compared with those of the ChR2 mutant E90Q. Based on the spectroscopic data we developed a model of the photocycle comprising six distinguishable intermediates. This photocycle shows similarities to the photocycle of the ChR2-related Channelrhodopsin of Volvox but also displays significant differences. We show that molecular changes include retinal isomerization, changes in hydrogen bonding of carboxylic acids, and large alterations of the protein backbone structure. These alterations are stronger than those observed in the photocycle of other microbial rhodopsins like bacteriorhodopsin and are related to those occurring in animal rhodopsins. UV-visible and Fourier transform infrared difference spectroscopy revealed two late intermediates with different time constants of τ = 6 and 40 s that exist during the recovery of the dark state. The carboxylic side chain of Glu90 is involved in the slow transition. The molecular changes during the ChR2 photocycle are discussed with respect to other members of the rhodopsin family.

Crystal structure of a common GPCR-binding interface for G protein and arrestin.

G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαβγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the ‘finger loop’ region (ArrFL1–4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins.

In Channelrhodopsin-2 Glu-90 Is Crucial for Ion Selectivity and Is Deprotonated during the Photocycle

Background: Channelrhodopsin-2 is a light-gated ion channel extensively used in optogenetics. Results: Glu-90 is deprotonated in the open state and is crucial for ion selectivity. Conclusion: Protonation change of Glu-90 is part of the opening/closing of the conductive pore, and the functional protein unit is assumed to be the monomer. Significance: Understanding the gating mechanism is necessary for optimizing this optogenetic tool. The light-activated microbial ion channel channelrhodopsin-2 (ChR2) is a powerful tool to study cellular processes with high spatiotemporal resolution in the emerging field of optogenetics. To customize the channel properties for optogenetic experiments, a detailed understanding of its molecular reaction mechanism is essential. Here, Glu-90, a key residue involved in the gating and selectivity mechanism of the ion channel is characterized in detail. The deprotonation of Glu-90 during the photocycle is elucidated by time-resolved FTIR spectroscopy, which seems to be part of the opening mechanism of the conductive pore. Furthermore, Glu-90 is crucial to ion selectivity as also revealed by mutation of this residue combined with voltage clamp experiments. By dynamic homology modeling, we further hypothesized that the conductive pore is flanked by Glu-90 and located between helices A, B, C, and G.

New infrared spectroscopic beamline at BESSY II

At BESSY a multipurpose infrared (IR) beamline is being built for biological and materials science investigations. It will provide useful IR intensities over the energy range from about 10 000 down to 50 cm−1 and lower. After commissioning the first beamline section in the spring of this year first quantitative measurements on the performance have been made in the near and mid-infrared wavelength region.

Spectroscopic studies and PM5 semiempirical calculations of new Schiff bases of gossypol with amino derivatives of crown ethers

Abstract Two Schiffs bases of racemic gossypol with amino derivatives of crown ethers were synthesised and studied by FT-IR and 1 H NMR spectroscopy, while their structures were calculated by the PM5 semiempirical method. These studied Schiffs bases exist in the solid state and in solutions as enamine forms. In the solid state the interactions between OH groups in the 6, 6′ positions and oxygen atoms of the crowns are dependent on the crown size whereas in solutions they are not. These interactions are very well evidenced in the FT-IR and 1 H NMR spectra. In the 1 H NMR spectra the existence of two enantiomers of the Schiffs bases are well visible. The structures of the Schiffs bases and the strength of the hydrogen bonds within these structures are discussed.

Synthesis and antimicrobial properties of monensin A esters.

The esters (2-10) of the ionophore antibiotic Monensin (1) were synthesized by four different methods, which are discussed in detail. These new esters were characterized by various spectroscopic techniques and subsequently tested in the face of their antimicrobial properties. Three derivatives (3, 8 and 10) showed activity against Gram-positive bacteria. Additionally derivative (10) exhibited a relatively low antifungal activity against Candida in contrast to Monensin A.

Homoconjugated hydrogen bonds with amidine and guanidine bases Osmometric, potentiometric and FTIR studies

Five very strong N bases, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), pK a =23.4; 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),pK a =23.9; tetramethylguanidine (TMG), pK a =23.3; 2-phenyl-tetramethylguanidine (PhTMG), pK a =20.6; and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), pK a =24.97; have been studied by osmometric measurements which showed that they are monomeric in acetonitrile solutions. The constants of the formation of homoconjugated complexes were determined by potentiometric measurements. In the IR spectra of the semi-protonated complexes of DBN, DBU and TMG, the homoconjugated N + –H···NN ···H– + N hydrogen bonds cause broad band complexes in the region 3200–2500 cm -1 instead of the expected continua. This spectral peculiarity is discussed.

Spectroscopic study and PM5 semiempirical calculations of tautomeric forms of gossypol Schiff base with n-butylamine in the solid state and in the solution

Abstract The Schiff base of gossypol with n -butylamine was synthesised and its structure was studied by FT-IR and 1 H NMR, 13 C NMR and 15 N NMR as well as PM5 semiempirical methods. It is shown that the Schiff base exists in solution and in solid state as the enamine–enamine tautomer. The structure of this tautomer is discussed in detail.

The branched photocycle of the slow-cycling channelrhodopsin-2 mutant C128T.

Channelrhodopsins (ChRs) of green algae such as Chlamydomonas are used as neuroscience tools to specifically depolarize cells with light. A crude model of the ChR2 photocycle has been recently established, but details of the photoreactions are widely unknown. Here, we present the photoreactions of a slow-cycling ChR2 mutant (step function rhodopsin), with C128 replaced by threonine and 200-fold extended lifetime of the conducting-state P520. At a late state of the photocycle, a fraction of the proteins branches off into an inactive species, P380, which accumulates during prolonged illumination. At neutral pH, P380 is converted into P353, a species with a characteristic fine-structured spectrum that is interpreted as retroretinyl chromophore. The described branching reactions should be considered, when ChR is used as a neuroscience tool, especially in the case of fluorescence imaging at high light intensities.

Early Formation of the Ion‐Conducting Pore in Channelrhodopsin‐2

Channelrhodopsins (ChRs) are light-gated ion channels that are widely used in optogenetics. They allow precise control of neuronal activity with light, but a detailed understanding of how the channel is gated and the ions are conducted is still lacking. The recent determination of the X-ray structural model in the closed state marks an important milestone. Herein the open state structure is presented and the early formation of the ion conducting pore is elucidated in atomic detail using time-resolved FTIR spectroscopy. Photo-isomerization of the retinal-chromophore causes a downward movement of the highly conserved E90, which opens the pore. Molecular dynamic (MD) simulations show that water molecules invade through this opened pore, Helix 2 tilts and the channel fully opens within ms. Since E90 is a highly conserved residue, the proposed E90-Helix2-tilt (EHT) model might describe a general activation mechanism and provides a new avenue for further mechanistic studies and engineering.