Recent advances in the photochemical control of protein function

Abstract

Biological processes are regulated with a high level of spatial and temporal resolution. To understand and manipulate these processes, scientists need to be able to regulate them with Nature's level of precision. In this context, light is a unique regulatory element because it can be precisely controlled in terms of location, timing and amplitude. Moreover, most biological laboratories have a wide range of light sources as standard equipment. This review article summarizes the most recent advances in light-mediated regulation of protein function and its application in a cellular context. Specifically, the photocaging of small-molecule modulators of protein function and of specific amino acid residues in proteins is discussed. In addition, examples of the photochemical control of protein function through the application of genetically engineered natural-light receptors are presented.

Figure 1

Four different approaches to control protein function with light. (a) Caged small effector molecules can inhibit protein function after caging group removal via UV irradiation. (b) Caged proteins, expressed using genetically encoded caged amino acids, can be activated via UV irradiation through caging group removal. (c) The activity of proteins can be reversibly regulated by light irradiation when fused to a light receptor (e.g. a natural light oxygen voltage (LOV) domain or a synthetic photoswitchable affinity label (PAL)). (d) Two proteins can be dimerized by light irradiation when fused to a natural plant phytochrome (e.g. PhyB) and a phytochrome interaction factor (PIF).

Figure 2

Light-activatable small molecule inhibitors of gene expression repressor proteins. (a) Caged IPTG 1. (b) Bacterial lithography experiment with 1, showing light-controlled induction and spatial control of β-galactosidase (left) and GFP (right) in a bacterial lawn (∅ 10 cm). (c) Caged doxycycline 2. (d) Spatial control of GFP expression using 2, with irradiation through a 344-µm photomask (scale bar = 250 µm); irradiated (blue) and non-irradiated (red) cells were quantified and fluorescence intensity is shown as a function of time. The light-removable caging groups are shown in red.

Figure 3

Reversible photochemical activation of a potassium ion (K⁺) channel. (a) Schematic of PAL-gated K⁺ channel. The sphere labeled with a plus sign represents a quaternary ammonium group, which blocks the channel when the diazobenzene is in the trans conformation. Irradiation at 380 nm switches the diazobenzene from trans to cis, thus enabling K⁺ flow. Irradiation with 500 nm light reverses the switching event, thus blocking the ion channel. (b) Light-controlled membrane potential of a PAL-gated K⁺ channel measured in dependence of light irradiation.

Figure 4

Photochemical control of protein localization in mammalian cells. (a) Genetically encoded caged lysine 3; light-removable caging group shown in red. (b) Gene diagram of p53-egfp, with the crucial lysine K305 bolded. (c) Nuclear import of EGFP in HEK293 cells after introduction and photolysis of 3 introduced into position 305 in NLS_p53. A mutation of K305 to tyrosine (K305→Y) blocks transport into the nucleus, regardless of light irradiation (left). Introduction of the caged lysine 3 at position 305 (K305→3) blocks transport of p53-EGFP-HA, but enables translocation into the nucleus after a brief light irradiation (365 nm, 5 s) (right). Scale bar = 10 µm.

Figure 5

Mechanism of LovTAP, a reversibly switchable DNA binding protein. (a) Before exposure to blue light, Jα (dark blue) is associated with the LOV domain (light blue), which renders the Trp repressor region (light orange) inactive. After irradiation with blue light (470 nm) a conformational change occurs and Jα (now dark orange) dissociates from the LOV domain, in turn activating the Trp repressor. The active protein binds to DNA at a lac operator region. The original conformation of the protein is resumed after incubation in the dark, and the Trp repressor region dissociates from the DNA, thus making the activity of LovTAP reversible. (b) LovTAP protects DNA against RsaI digestion at the lac operator site. Increasing the concentration of LovTAP (0–2.8 µM) decreases digestion, as does irradiation with blue light (dashed lines: irradiated; solid lines: non-irradiated).

Figure 6

Light-controlled expression of LacZ by PhyB-PIF protein dimerization. (a) Schematic of the light-controlled transcriptional activator. Phytochrome (Phy), which is fused to the DNA-binding region of GAL4 (GBD), reversibly binds to PIF. PIF is fused to the transcriptional activating domain of GAL4 (GAD), thus activating gene expression upon exposure to red light and ceasing activity by dissociation upon exposure to IR light. (b) LacZ (β-galactosidase) activity induced with pulses of red light (Rp, black line), and arrested with pulses of IR light (FRp, red line).