Thermoresponsive Microgel-Based Free-Standing Membranes: Influence of Different Microgel Cross-Linkers on Membrane Function.

In this study we show a possibility to produce thermoresponsive, free-standing microgel membranes based on N-isopropylacrylamide (NIPAM) and the UV-sensitive comonomer 2-hydroxy-4-(methacryloyloxy)benzophenone (HMABP). To influence the final network structure and functionality of the membranes, we use different cross-linkers in the microgel syntheses and characterize the resulting structural microgel properties and the swelling behavior by means of AFM, FTIR, and PCS measurements. Varying the cross-linker results in significant changes in the structure and swelling behavior of the individual microgels and has an influence on the incorporation of the comonomer, which is essential for subsequent photochemical membrane formation. We investigate the ion transport through the different membranes by temperature-dependent resistance measurements revealing a sharp increase in resistance when the copolymer microgels reach their collapsed state. The resistance of the membranes can be adjusted by different cross-linkers and the associated incorporation of the comonomer. Furthermore, we show that transferring a reversible cross-linker from a cross-linked state to an un-cross-linked state strongly influences the membrane properties and even reverses the switching behavior, while the mechanical stability of the membrane is maintained.

In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells.

Proteins are usually studied in well-defined buffer conditions, which differ substantially from those within a host cell. In some cases, the intracellular environment has an impact on the mechanism, which might be missed by in vitro experiments. IR difference spectroscopy previously has been applied to study the light-induced response of photoreceptors and photoenzymes in vitro Here, we established the in-cell IR difference (ICIRD) spectroscopy in the transmission and attenuated total reflection configuration to investigate the light-induced response of soluble proteins in living bacterial cells. ICIRD spectroscopy on the light, oxygen, or voltage (LOV) domains of the blue light receptors aureochrome and phototropin revealed a suppression of the response of specific secondary structure elements, indicating that the intracellular environment affects LOV photoreceptor mechanisms in general. Moreover, in-cell fluorescence spectroscopy disclosed that the intracellular environment slows down the recovery of the light-induced flavin adduct. Segment-resolved ICIRD spectroscopy on basic-region leucine zipper (bZIP)-LOV of aureochrome 1a from the diatom Phaeodactylum tricornutum indicated a signal progression from the LOV sensor to the bZIP effector independent of unfolding of the connecting A'α-helix, an observation that stood in contrast to in vitro results. This deviation was recapitulated in vitro by emulating the intracellular environment through the addition of the crowding agent BSA, but not by sucrose polymers. We conclude that ICIRD spectroscopy is a noninvasive, label-free approach for assessing conformational changes in receptors in living cells at ambient conditions. As demonstrated, these near-native responses may deviate from the mechanisms established under in vitro conditions.

Tongue Refolding in the Knotless Cyanobacterial Phytochrome All2699.

Phytochromes regulate central responses of plants and microorganisms such as shade avoidance and photosystem synthesis. Canonical phytochromes comprise a photosensory module of three domains. The C-terminal phytochrome-specific (PHY) domain interacts via a tongue element with the bilin chromophore in the central GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain. The bilin isomerizes upon illumination with red light, transforming the receptor from the Pr state to the Pfr state. The "knotless" phytochrome All2699 from the cyanobacterium Nostoc sp. PCC7120 comprises three GAF domains as a sensory module and a histidine kinase as an effector. GAF1 and GAF3 both bind a bilin, and GAF2 contains a tongue-like element. We studied the response of All2699, GAF1-GAF2, and GAF1 to red light by Fourier transform infrared difference spectroscopy, including a 13C-labeled protein moiety for assignment. In GAF1-GAF2, a refolding of the tongue from ß-sheet to α-helix and an upshift of the ring D carbonyl stretch from 1700 to 1712 cm-1 were observed. Therefore, GAF1-GAF2 is regarded as the smallest model system available to study the tongue response and interaction with the chromophore. Replacement of an arginine in the tongue with proline (R387P) did not affect the unfolding of the ß-sheet to Pfr but strongly impaired α-helix formation. In contrast, the Y55H mutation close to bilin ring D did not interfere with conversion to Pfr. Strikingly, the presence of GAF3 in the full-length All2699 diminished the response of the tongue and generated the signal pattern found for GAF1 alone. These results point to a regulatory or integrative role of GAF3 in All2699 that is absent in canonical phytochromes.

Biphasic Formation of 2D Nanomembranes by Photopolymerization of Diacetylene Lipids as Revealed by Infrared Difference Spectroscopy.

Two-dimensional nanomembranes are promising materials for filtration or separation by providing the basis for controlled and rapid transport between two compartments. The polymerization by UV light of diacetylene-containing lipids at an interface produces free-standing 2D nanomembranes. Here, we analyzed in situ the nanomembrane formation of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1-palmitoyl-2-(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (PTPE) on germanium using light-induced infrared difference spectroscopy with attenuated total reflection to obtain insights into the kinetics and mechanism of the polymerization process. Our interpretation is supported by atomic force microscopy and density functional theory. Formation of the polymer network is evidenced by changes in the frequency of C═O stretches acting as infrared probes. However, spectral and kinetic analysis revealed a biphasic process in the monolayer. In both phases, losses in signal of CH2 stretches are observed which are not in agreement with the accepted mechanism of chain propagation for diacetylene polymerization. These signals are dominant in the second phase and are assigned to termination reactions with some contributions from intramolecular consecutive reactions. This finding now provides a spectroscopic measure for the identity and integrity of the nanomembrane complementary to microscopic analysis. We deduce that limited 2D mobility on the solid support promotes intramolecular termination, leading to smaller domains.

Characterization of Robust and Free-Standing 2D-Nanomembranes of UV-Polymerized Diacetylene Lipids.

Free-standing lipid membranes are promising as artificial functional membrane systems for application in separation, filtration, and nanopore sensing. To improve the mechanical properties of lipid membranes, UV-polymerized lipids have been introduced. We investigated free-standing as well as substrate-supported monolayers of 1-palmitoyl-2-(10,12-tricosadiynoyl)- sn-glycero-3-phosphoethanolamine (PTPE) and 1,2-bis(10,12-tricosadiynoyl)- sn-glycero-3-phosphocholine (DiynePC) and characterized them with respect to their structure, morphology, and stability. Using helium ion microscopy (HIM), we were able to visualize the integrity of the lipid 2D-nanomembranes spanning micrometer-sized voids under high-vacuum conditions. Atomic force microscopy (AFM) investigations under ambient conditions revealed formation of intact and robust pore-spanning 2D-nanomembranes up to 8 × 2 µm2 in size. Analysis by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) verified a distinct reduction of signal at 2143 cm-1 from diacetylene groups in the 2D-nanomembranes after UV-polymerization. Further high-resolution AFM investigations of unpolymerized lipid monolayers revealed a well-ordered two-dimensional network, when deposited on highly oriented pyrolytic graphite (HOPG). These structures were inhibited for polymerized adlayers. Structural models for the molecular arrangement of the adlayers are proposed and discussed.

Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates.

pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK' = 6.6) and EGF-CoGFP-mCRISPRed (pK' = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein-drug conjugates in cancer therapy.

Plant Cryptochromes Illuminated: A Spectroscopic Perspective on the Mechanism.

Plant cryptochromes are central blue light receptors for the control of land plant and algal development including the circadian clock and the cell cycle. Cryptochromes share a photolyase homology region with about 500 amino acids and bind the chromophore flavin adenine dinucleotide. Characteristic for plant cryptochromes is a conserved aspartic acid close to flavin and an exceptionally long C-terminal extension. The mechanism of activation by excitation and reduction of the chromophore flavin adenine dinucleotide has been controversially discussed for many years. Various spectroscopic techniques have contributed to our understanding of plant cryptochromes by providing high time resolution, ambient conditions and even in-cell approaches. As a result, unifying and differing aspects of photoreaction and signal propagation have been revealed in comparison to members from other cryptochrome subfamilies. Here, we review the insight from spectroscopy on the flavin photoreaction in plant cryptochromes and present the current models on the signal propagation from flavin reduction to dissociation of the C-terminal extension.

Resolving Structural Changes of Photoreceptors in Living Escherichia coli via In-cell Infrared Difference Spectroscopy.

Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions in vivo differ dramatically from those selected for in vitro experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or binding of small molecules. Fourier transform infrared (FTIR) difference spectroscopy is a well-suited method to study the light-induced structural responses of photoreceptors including changes in cofactor, side chains and secondary structure. Here, we describe a protocol to study the response of cofactor and protein in living E. coli cells via in-cell infrared difference (ICIRD) spectroscopy using the attenuated total reflection (ATR) configuration. Proteins are overexpressed in E. coli, the cells are transferred into saline solution and the copy number per cell is determined using fluorescence spectroscopy. The suspension is centrifuged and the concentrated cells transferred onto the ATR cell inside the FTIR spectrometer. The thermostatted cell is sealed and illuminated from the top with an LED. Intensity spectra are recorded before and after illumination to generate the difference spectrum of the receptor inside the living cell. With ICIRD spectroscopy, structural changes of soluble photoreceptors are resolved in a near-native environment. The approach works in H2O at ambient conditions, is label free, without any limitations in protein size and does not require any purification step. Graphic abstract: In-cell infrared difference spectroscopy on photoreceptors in living E. coli using attenuated total reflection.

C-Terminal Extension of a Plant Cryptochrome Dissociates from the ß-Sheet of the Flavin-Binding Domain.

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from Chlamydomonas reinhardtii in living bacterial cells, because the receptor degraded upon isolation. We demonstrate a stabilization of the flavin neutral radical as photoproduct and of the resulting ß-sheet reorganization by binding of cellular ATP. Comparison between light-induced structural responses of full-length pCRY and PHR reveals a downshift in frequency of the ß-sheet signal, implying an association of the CCT close to the only ß-sheet of the PHR in the dark. We provide a missing link in activation of plant cryptochromes after flavin photoreduction by indicating that ß-sheet reorganization causes the CCT release and restructuring.

Aurophilicity in action: stepwise formation of dinuclear Au(i) macrocycles with rigid 1,8-dialkynylanthracenes.

Two phosphane-gold(i) functions (with tri(para-tolyl)- or tri-n-butylphosphane) were attached to a 1,8-diethynylanthracene backbone. The ligand size prevents direct interaction between the gold atoms, but the initial products rearrange to form macrocyclic anions comprising two 1,8-diethynylanthracene and two gold units, linked to cationic gold(i) fragments by maximising aurophilic interactions.