Using photocaging for fast time-resolved structural biology studies

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 (ϵ_max 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.

Keywords: photocages; reaction initiation; serial crystallography; time-resolved structural biology.

Figure 1

Achievable time resolutions for different protein-activation methods (Levantino, Yorke et al., 2015 ▸). Different protein transitions are depicted along with their typical timescales and length scales. X-ray crystallo­graphy, small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) can capture different types of structural transitions. Direct laser excitation, temperature jumps and mixing are the usual approaches to sample triggering and synchronization. The typical decaging half-lives of the four main photocaging groups addressed in this review (coumarin, p-hydroxyphenyl, o-nitrobenzyl and nitrodi­benzofuran) are shown as black bars.

Figure 2

Examples of biologically relevant photocaged small molecules for which rates of photo-decaging have been reported. The compounds are ordered so as to facilitate comparison between similar scaffolds. Color-coding indicates compound classes: o-nitrobenzyl (red), red-shifted o-nitrobenzyl (orange), nitrodibenzylfuranyl (yellow), p-hydroxyphenyl (green) and coumarinyl (blue). Each plot gives values for five properties: maximum absorption wavelength (λ_max), extinction coefficient at λ_max (ɛ_max), cleavage rate, quantum yield (φ) and a qualitative assessment of the expected solubility. The photo-released groups are highlighted in bold. The point of photocage attachment for each compound is phosphate (ATP), γ-carboxylate or amine (Glu), or ether (EGTA), as shown in Fig. 3 ▸.

Figure 3

Full representation of the photocaged compounds shown in Fig. 2 ▸. The covalent bonds for attachment of the photocage to the small molecule and its release are highlighted by cleavage lines.

Figure 4

The chemistry of photocleavage. Upon illumination, the photocages undergo a ‘light transition’ into an excited state. Nonproductive events, such as fluorescence or nonradiative decay, can bring the molecule back to the ground state, greatly decreasing the quantum yield of cleavage. The dark reaction proceeds from the excited state through one or more intermediates. The dark reaction determines the rate of compound release and therefore the achievable time resolution.

Figure 5

The correlation between sample thickness, concentration and extinction coefficient and their effect on light transmission. The figure shows the 50%, 75% and 90% transmission thresholds of light through samples of varying thickness (10 µm to 1 mm, color-coded). These transmission thresholds correspond to 50%, 25% and 10% attenuation, respectively. The extinction coefficient (x axis) and sample concentration (y axis) are varied. Insets (a)–(c) show different areas in more detail for the 75% transmission plot, where (a) is high ɛ and low concentration, (b) is low ɛ and high concentration and (c) is low ɛ and low concentration.

Figure 6

Cobalt-based O₂ photocage.