Stabilization of a flavoprotein for solid-state photo-CIDNP MAS NMR at room temperature by embedding in a glassy sugar matrix

Red Shift in the Absorption Spectrum of Phototropin LOV1 upon the Formation of a Semiquinone Radical: Reconstructing the Orbital Architecture

“…First, a sign change of FMN-N10 and W98-N1 from negative, i.e., emissive (frozen solution), to positive, i.e., enhanced absorptive (trehalose glass), occurs. This seems to be a feature of trehalose-embedded Cr LOV1, as has been shown earlier, and has been speculated to be related to the shortening of the SCRP lifetime affecting the spin dynamics . For example, in a trehalose-embedded photosynthetic reaction center, radical pair recombination is reduced by a factor of 2 compared to a water-glycerol matrix .…”

“…Trehalose glass provides a unique matrix for spectroscopic investigations of proteins at room temperature. Its ability to stabilize the protein backbone and simultaneously keep dynamic processes intact, albeit at a reduced rate, in conjunction with its limited light scattering characteristic, can be utilized to study photodynamic processes such as electron transfer or photoswitching. , Cr LOV1 incorporated into trehalose glass is a prime candidate for NMR studies, exploiting the solid-state photo-CIDNP effect and creating hyperpolarized signals. Our earlier study demonstrated its feasibility to study hyperpolarized 13 C signals in a flavoprotein at natural abundance.…”

“…Its ability to stabilize the protein backbone and simultaneously keep dynamic processes intact, albeit at a reduced rate, in conjunction with its limited light scattering characteristic, can be utilized to study photodynamic processes such as electron transfer or photoswitching. , Cr LOV1 incorporated into trehalose glass is a prime candidate for NMR studies, exploiting the solid-state photo-CIDNP effect and creating hyperpolarized signals. Our earlier study demonstrated its feasibility to study hyperpolarized 13 C signals in a flavoprotein at natural abundance. In the present study, we show solid-state photo-CIDNP MAS NMR spectra obtained from uniformly 15 N enriched Cr LOV1 in both a frozen buffer solution and a solid trehalose matrix (see Figure ).…”

“…During light irradiation, the surrounding protein undergoes photodegradation due to the formation of singlet oxygen. − Recently, stabilization and protection of flavin-containing proteins by embedding in an amorphous matrix of the disaccharide trehalose has been reported . The sugar matrix still allowed for observation of photo-CIDNP enhancement in solids at room temperature while prolonging the effective measurement time significantly from minutes to hours.…”

“… 18 − 21 Recently, stabilization and protection of flavin-containing proteins by embedding in an amorphous matrix of the disaccharide trehalose has been reported. 22 The sugar matrix still allowed for observation of photo-CIDNP enhancement in solids at room temperature while prolonging the effective measurement time significantly from minutes to hours. It has been demonstrated that incorporation into trehalose matrices dramatically slows down processes such as electron transfer reactions 23 , 24 or light-induced chromophore changes, 25 while maintaining the protein structure.…”