Abstract
Careful selection of photocaging approaches is critical to achieve fast and well synchronized reaction initiation and perform successful time-resolved structural biology experiments. This review summarizes the best characterized and most relevant photocaging groups previously described in the literature. It also provides a walkthrough of the essential factors to consider in designing a suitable photocaged molecule to address specific biological questions, focusing on photocaging groups with well characterized spectroscopic properties. The relationships between decay rates (k in s-1), quantum yields (ϕ) and molar extinction coefficients (ϵmax in M-1 cm-1) are highlighted for different groups. The effects of the nature of the photocaged group on these properties is also discussed. Four main photocaging scaffolds are presented in detail, o-nitrobenzyls, p-hydroxyphenyls, coumarinyls and nitrodibenzofuranyls, along with three examples of the use of this technology. Furthermore, a subset of specialty photocages are highlighted: photoacids, molecular photoswitches and metal-containing photocages. These extend the range of photocaging approaches by, for example, controlling pH or generating conformationally locked molecules.
Highlights
The timescales of interest in biomolecular science span a wide range, from local reaction chemistry occurring on femtosecond (10À15 s) to nanosecond (10À9 s) timescales to longrange motions occurring over much slower timescales
The rapid development of novel sampledelivery methods (Cheng, 2020; Grunbein & Nass Kovacs, 2019) has accompanied these advances, providing platforms for the fast sample refreshment which is needed for serial crystallography experiments. Such platforms utilize clever setups such as X-ray-compatible fixed targets (Schulz et al, 2018; Roedig et al, 2016) and enclosed microfluidics (Tosha et al, 2017; Sui & Perry, 2017; Monteiro et al, 2019, 2020) as well as liquid jets (Martiel et al, 2019) and viscous jets (Grunbein & Nass Kovacs, 2019; Martin-Garcia et al, 2017). All of these technological developments have led to a boost in interest in time-resolved structural biology, and a rapid increase in the number of systems that could be studied over the last decade
As this review focuses on the use of photocages for fast time-resolved structural biology experiments, the following discussion will focus on compounds for which rates of cleavage have been determined
Summary
The timescales of interest in biomolecular science span a wide range, from local reaction chemistry occurring on femtosecond (10À15 s) to nanosecond (10À9 s) timescales to longrange motions (changes in macromolecular conformation) occurring over much slower timescales (tens of milliseconds to seconds; Fig. 1). Such platforms utilize clever setups such as X-ray-compatible fixed targets (Schulz et al, 2018; Roedig et al, 2016) and enclosed microfluidics (Tosha et al, 2017; Sui & Perry, 2017; Monteiro et al, 2019, 2020) as well as liquid jets (Martiel et al, 2019) and viscous jets (Grunbein & Nass Kovacs, 2019; Martin-Garcia et al, 2017) All of these technological developments have led to a boost in interest in time-resolved structural biology, and a rapid increase in the number of systems that could be studied over the last decade. The macromolecules in the crystal or solution samples have to be synchronized in order to obtain a clear picture of the structural changes, and the reaction must be triggered uniformly through the sample on a timescale that is commensurate with the reaction steps of interest
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