Spiropyran labeling for sensitive probing of protein diffusion by the transient grating method

Time-resolved detection of SDS-induced conformational changes in α-synuclein by a micro-stopped-flow system

“…NSP was synthesized as reported previously. 31 DSP was synthesized by the coupling reaction of SP-COOH and N-(2-aminoethyl)maleimide with condensation reagent 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC). SP-COOH, 1-(bcarboxyethyl)-3,3-dimethyl-6-nitrospiro(indoline-2,2(2H-1)benzopyran) (57 mmol) was activated with EDC (68 mmol) in the presence of 3H-1,2,3-triazolo[4,5-b]pyridin-3-ol (68 mmol) in 0.8 mL DMF for 10 min at 25 C. To this solution, N-(2-aminoethyl)maleimide (57 mmol) in 0.2 mL DMF was added and reacted for 10 min at 25 C. The reactant was evaporated, and DSP was puried by ash column chromatography packed using a silica gel in a solvent of 20% methanol and 80% chloroform.…”

“…Furthermore, the probe molecule should give rise to a strong TG signal. In our previous work, we have reported a candidate for such a purpose, that is, one of the spiropyran 30 derivatives, 31 In this work, for site-specic labeling of aSyn, another type of spiropyran derivative, 3-…”

“…SI-1A † ). 31 In this work, for site-specific labeling of αSyn, another type of spiropyran derivative, 3-{3′,3′-dimethyl-6-nitrospiro[chromene-2,2′-indole]-1′-yl}- N -[2-(2,5-dioxopyrrol-1yl)ethyl]propanamide (DSP) was used. Upon photoexcitation of these dyes at 308 nm, the ring-closed spiropyran (SP) form is converted to the ring-opened merocyanine (MC) form, which gives rise to a strong TG signal.…”

A Time-Resolved Diffusion Technique for Detection of the Conformational Changes and Molecular Assembly/Disassembly Processes of Biomolecules

“…If the molecule is photoreactive, such as a photosensor protein, it is straightforward to obtain the TG signal, but if not, it is necessary to label the protein with a photoreactive molecule. For example, when a protein is labeled with a photochromic probe molecule, the light-induced change in the absorption spectrum creates the spatial modulation of the refractive index, which acts as a grating to produce the TG signal ( Eitoku and Terazima, 2008 ; Takaramoto et al, 2020a ). The diffracted light intensity then decays due to the diffusion.…”

“…For the TG measurement, the C-terminal end of αSyn was labeled with a small photochromic molecule of spiropyran ( Klajn, 2014 ; Takaramoto et al, 2020a , b ). Upon photoexcitation at 308 nm, the colorless ring-closed spiropyran (SP) form is converted to the violet-colored ring-opened merocyanine (MC) form, which causes a strong TG signal.…”