Conformational and Intermolecular Interaction Dynamics of Photolyase/Cryptochrome Proteins Monitored by the Time‐Resolved Diffusion Technique

Cryptochrome (CRY), a blue light sensor protein, possesses a similar domain structure to photolyase (PHR) that, upon absorption of light, repairs DNA damage. In this review, we compare the reaction dynamics of these systems by monitoring the reaction kinetics of conformational change and intermolecular interaction change based on time‐dependent diffusion coefficient measurements obtained by using the pulsed laser‐induced transient grating technique. Using this method, time‐dependent biomolecular interactions, such as transient dissociation reactions in solution, have been successfully detected in real time. Conformational change in (6‐4) PHR has not been detected after the photoexcitation by monitoring the diffusion coefficient. However, the repaired DNA dissociates from PHR with a time constant of 50 μs, which must relate to a minor conformational change. However, CRY exhibits a considerable diffusion change with a time constant of 400 ms, which indicates that the protein–solvent interaction is changed by the conformational change. The C‐terminal domain of CRY is shown to be responsible for this change.     

Ionization‐Induced Solvent Migration in Acetanilide‐Methanol Clusters Inferred from Isomer‐Selective Infrared Spectroscopy

We present the resonance‐enhanced multiphoton ionization, infrared‐ultraviolet hole burning (IR‐UV HB), and IR dip spectra of the trans‐acetanilide–methanol (AA–MeOH) cluster in the S₀, S₁, and cationic ground state (D₀) in a supersonic jet. The IR‐UV HB spectra demonstrate the co‐existence of two isomers in S_0,1, in which MeOH binds either to the NH or the CO site of the peptide linkage in AA, denoted as AA(NH)–MeOH and AA(CO)–MeOH. When AA(CO)–MeOH is selectively ionized, its IR spectrum in D₀ is the same as that measured for AA⁺(NH)–MeOH. Thus, photoionization of AA(CO)–MeOH induces migration of MeOH from the CO to the NH site with 100% yield.     

Ligand‐Induced Conformational Changes in HSP90 Monitored Time Resolved and Label Free—Towards a Conformational Activity Screening for Drug Discovery

Investigation of protein–ligand interactions is crucial during early drug‐discovery processes. ATR‐FTIR spectroscopy can detect label‐free protein–ligand interactions with high spatiotemporal resolution. Here we immobilized, as an example, the heat shock protein HSP90 on an ATR crystal. This protein is an important molecular target for drugs against several diseases including cancer. With our novel approach we investigated a ligand‐induced secondary structural change. Two specific binding modes of 19 drug‐like compounds were analyzed. Different binding modes can lead to different efficacy and specificity of different drugs. In addition, the k_obs values of ligand dissociation were obtained. The results were validated by X‐ray crystallography for the structural change and by SPR experiments for the dissociation kinetics, but our method yields all data in a single and simple experiment.     

Near‐Infrared Light‐Initiated Hybridization Chain Reaction for Spatially and Temporally Resolved Signal Amplification

Precise control over signal amplification provides unparalleled opportunities for diverse applications. However, spatiotemporally controlled amplification has not been realized because of the lack of a design methodology. The aim of this study was thus to develop a conceptual approach for remote control over signal amplification at a chosen time and site in living cells. This system was constructed by re‐engineering the functional units of the hybridization chain reaction (HCR) and combination with upconversion photochemistry, thus resulting in an activatable HCR with the high spatial and temporal precision of near‐infrared (NIR) light. As a proof of concept, we demonstrate the spatially and temporally resolved amplified imaging of messenger RNA (mRNA) with ultrahigh sensitivity in vitro and in vivo. Furthermore, by using a system targeting subcellular sites we have developed a new technique for NIR‐initiated amplified imaging of mRNA exclusively within a specific organelle.     

Sol‐to‐Gel Transition in Fast Evaporating Systems Observed by in Situ Time‐Resolved Infrared Spectroscopy

The in situ observation of a sol‐to‐gel transition in fast evaporating systems is a challenging task and the lack of a suitable experimental design, which includes the chemistry and the analytical method, has limited the observations. We synthesise an acidic sol, employing only tetraethylorthosilicate, SiCl₄ as catalyst and deuterated water; the absence of water added to the sol allows us to follow the absorption from the external environment and the evaporation of deuterated water. The time‐resolved data, obtained by attenuated total reflection infrared spectroscopy on an evaporating droplet, enables us to identify four different stages during evaporation. They are linked to specific hydrolysis and condensation rates that affect the uptake of water from external environment. The second stage is characterized by a decrease in hydroxyl content, a fast rise of condensation rate and an almost stationary absorption of water. This stage has been associated with the sol‐to‐gel transition.     

Nuclear‐Spin‐Induced Circular Dichroism in the Infrared Region for Liquids

Recently, the nuclear‐spin‐induced optical rotation (NSOR) and circular dichroism (NSCD) for liquids were discovered and extensively studied and developed. However, so far, nuclear‐spin‐induced magnetic circular dichroism in the IR region (IR‐NSCD) has not been explored, even though all polyatomic molecules exhibit extensive IR spectra. Herein, IR‐NSCD is proposed and discussed theoretically. The results indicate that in favorable conditions the IR‐NSCD angle may be much larger than the NSOR angle in the UV/Vis region due to a vibrational resonance effect and can be measurable by using the NSOR experiment scheme. IR‐NSCD can automatically combine and give NMR spectra and IRCD spectra of the nuclear spin prepolarized samples in liquids, which, in principle, could be developed to become a unique, novel analytical tool.     

Visible‐Light‐Induced Selective Defluoroborylation of Polyfluoroarenes,gem‐Difluoroalkenes,and Trifluoromethylalkenes

Fluorinated organoboranes serve as versatile synthetic precursors for the preparation of value‐added fluorinated organic compounds. Recent progress has been mainly focused on the transition‐metal catalyzed defluoroborylation. Herein, we report a photocatalytic defluoroborylation platform through direct B?H activation of N‐heterocyclic carbene boranes, through the synergistic merger of a photoredox catalyst and a hydrogen atom transfer catalyst. This atom‐economic and operationally simple protocol has enabled defluoroborylation of an extremely broad scope of multifluorinated substrates including polyfluoroarenes, gem‐difluoroalkenes, and trifluoromethylalkenes in a highly selective fashion. Intriguingly, the defluoroborylation protocol can be transition‐metal free, and the regioselectivity obtained is complementary to the reported transition‐metal‐catalysis in many cases.     

Visible‐Light‐Induced Morphological Changes of Giant Vesicles by Photoisomerization of a Ruthenium Aqua Complex

Visible‐ and red‐light responsive vesicles were prepared by incorporating a ruthenium aqua complex having two alkyl chains on tridentate and asymmetrical bidentate ligands (proximal‐ 2 : [Ru(C₁₀tpy)(C₁₀pyqu)OH₂]²⁺, C₁₀tpy=4′‐decyloxy‐2,2′;6′,2“‐terpyridine, C₁₀pyqu=2‐[2′‐(6′‐decyloxy)‐pyridyl]quinoline). The ruthenium complex of proximal‐ 2 with closed alkyl chain geometry and a cylinder‐like molecular shape exhibited photoisomerization to distal‐ 2 with an open alkyl chain geometry and a cone‐like shape, both in an aqueous solution and in vesicle dispersions. We observed that light irradiation of giant vesicles containing proximal‐ 2 induced diverse morphological changes.     

Real‐Time Band‐Selective Homonuclear Proton Decoupling for Improving Sensitivity and Resolution in Phase‐Sensitive J‐Resolved Spectroscopy

Real‐time band‐selective homonuclear ¹H decoupling during data acquisition of z‐filtered J‐resolved spectroscopy produces ¹H‐decoupled ¹H NMR spectra and leads to sensitivity enhancement and improved resolution, and thus aids the measurement of J couplings and residual dipolar couplings in crowded regions of ¹H NMR spectrum. High quality spectra from peptides, organic molecules, and also from enantiomers dissolved in weakly aligned chiral media are reported.     

Direct Observation of Aggregation‐Induced Backbone Conformational Changes in Tau Peptides

In tau proteins, the hexapeptides in the R2 and R3 repeats are known to initiate tau fibril formation, which causes a class of neurodegenerative diseases called the taupathies. We show that in R3, in addition to the presence of the hexapeptides, the correct turn conformation upstream to it is also essential for producing prion‐like fibrils that are capable of propagation. A time‐dependent NMR aggregation assay of a slow fibril forming R3‐S316P peptide revealed a trans to cis equilibrium shift in the peptide‐bond conformation preceding P316 during the growth phase of the aggregation process. S316 was identified as the key residue in the turn that confers templating capacity on R3 fibrils to accelerate the aggregation of the R3‐S316P peptide. These results on the specific interactions and conformational changes responsible for tau aggregation could prove useful for developing an efficient therapeutic intervention in Alzheimer's disease.     

Conformational Relaxation of p‐Phenylenevinylene Trimers in Solution Studied by Picosecond Time‐Resolved Fluorescence

Two p‐phenylenevinylene (PV) trimers, containing 3′‐methylbutyloxyl (in MBOPV3) and 2′‐ethylhexyloxyl (in EHOPV3) side chains, are used as model compounds of PV‐based conjugated polymers (PPV) with the purpose of clarifying the origin of fast (picosecond time) components observed in the fluorescence decays of poly[2‐methoxy‐5‐(2′‐ethylhexyloxy)‐p‐phenylenevinylene] (MEH‐PPV). The fluorescence decays of MBOPV3 and EHOPV3 reveal the presence of similar fast components, which are assigned to excited‐state conformational relaxation of the initial population of non‐planar trimer conformers to lower‐energy, more planar conformers. The rate constant of conformational relaxation k_CR is dependent on solvent viscosity and temperature, according to the empirical relationship k_CR=aη_o^?α?exp(?αE_η/RT), where aη_o^?α is the frequency factor, η_o is the pre‐exponential coefficient of viscosity, E_η is the activation energy of viscous flow. The empirical parameter α, relating the solvent microscopic friction involved in the conformational change to the macroscopic solvent friction (α=1), depends on the side chain. The fast component in the fluorescence decays of MEH‐PPV polymers (PPVs), is assigned to resonance energy transfer from short to longer polymer segments. The present results call for revising this assignment/interpretation to account for the occurrence of conformational relaxation, concurrently with energy transfer, in PPVs.     

Tuning and Erasing Surface Wrinkles by Reversible Visible‐Light‐Induced Photoisomerization

Periodic wrinkling across different scales has received considerable attention because it not only represents structure failure but also finds wide applications. How to prevent wrinkling or create desired wrinkling patterns is non‐trivial because the dynamic evolution of wrinkles is a highly nonlinear problem. Herein, we report a simple yet powerful method to dynamically tune and/or erase wrinkling patterns with visible light. The light‐induced photoisomerization of azobenzene units in azopolymer films leads to stress release and consequently to the erasure of the wrinkles. The wrinkles in unexposed regions are also affected and oriented perpendicular to the exposed boundary during the stress reorganization. Theoretical models were developed to understand the dynamics of the reversible photoisomerization‐induced wrinkle evolution. This method can be applied for designing functional materials/devices, for example, for the reversible optical writing/erasure of information as demonstrated here.     

Pump‐Probe Fragmentation Action Spectroscopy: A Powerful Tool to Unravel Light‐Induced Processes in Molecular Photocatalysts

We present a proof of concept that ultrafast dynamics combined with photochemical stability information of molecular photocatalysts can be acquired by electrospray ionization mass spectrometry combined with time‐resolved femtosecond laser spectroscopy in an ion trap. This pump‐probe “fragmentation action spectroscopy” gives straightforward access to information that usually requires high purity compounds and great experimental efforts. Results of gas‐phase studies on the electronic dynamics of two supramolecular photocatalysts compare well to previous findings in solution and give further evidence for a directed electron transfer, a key process for photocatalytic hydrogen generation.     

A Light‐Up Probe with Aggregation‐Induced Emission for Real‐Time Bio‐orthogonal Tumor Labeling and Image‐Guided Photodynamic Therapy

Bio‐orthogonal tumor labeling is more effective in delivering imaging agents or drugs to a tumor site than active targeting strategy owing to covalent ligation. However, to date, tumor‐specific imaging through bio‐orthogonal labeling largely relies on body clearance to differentiate target from the intrinsic probe signal owing to the lack of light‐up probes for in vivo bio‐orthogonal labeling. Now the first light‐up probe based on a fluorogen with aggregation‐induced emission for in vivo bio‐orthogonal fluorescence turn‐on tumor labeling is presented. The probe has low background fluorescence in aqueous media, showing negligible non‐specific interaction with normal tissues. Once it reacts with azide groups introduced to tumor cells through metabolic engineering, the probe fluorescence is lightened up very quickly, enabling rapid tumor‐specific imaging. The photosensitizing ability was also used to realize effective image‐guided photodynamic tumor therapy.     

Monitoring Conformational Changes in the NDM‐1 Metallo‐β‐lactamase by 19F NMR Spectroscopy

The New Delhi metallo‐β‐lactamase (NDM‐1) is involved in the emerging antibiotic resistance problem. Development of metallo‐β‐lactamases (MBLs) inhibitors has proven challenging, due to their conformational flexibility. Here we report site‐selective labeling of NDM‐1 with 1,1,1‐trifluoro‐3‐bromo acetone (BFA), and its use to study binding events and conformational changes upon ligand–metal binding using ¹⁹F NMR spectroscopy. The results demonstrate different modes of binding of known NDM‐1 inhibitors, including L ‐ and D ‐captopril by monitoring the changing chemical environment of the active‐site loop of NDM‐1. The method described will be applicable to other MBLs and more generally to monitoring ligand‐induced conformational changes.