Dwelling in the dark: procedures for the crystallography of phytochromes and other photochromic proteins

Abstract

Crystallization of phytochromes and other photochromic proteins is hampered by the conformational changes that they undergo on exposure to light. As a canonical phytochrome, cyanobacterial Cph1 switches between two stable states upon absorption of red/far-red light. Consequently, it is mandatory to work in darkness from protein purification to crystal cryoprotection in order to ensure complete occupancy of one state or the other. With the simple and inexpensive methods that have been developed, phytochromes and other photochromic molecules can effectively be handled and crystallized, as has been demonstrated by the solution of the three-dimensional structure of the Cph1 sensory module.

Structure of the Cyanobacterial Phytochrome 2 Photosensor Implies a Tryptophan Switch for Phytochrome Signaling

“…These data are in line with previous observations on the photosensory module of SynCph1, where long x-ray radiation induced additional absorbance peaks in the UV-visible spectra (43). In SynCph2(1-2) the x-ray-induced species relaxes in at (1-2).…”

Spectroscopy and a High-Resolution Crystal Structure of Tyr263 Mutants of Cyanobacterial Phytochrome Cph1

“…10 The protein was expressed as described previously. 13,61 For dimerization studies, SEC was performed with a red-irradiated 3 mg/ml sample using a 16/60 Superdex 200 column (GE Healthcare; V t = 120.6 ml, V 0 = 39.8 ml and flow rate = 1 ml/min) in 50 mM Tris-HCl (pH 7.8), 1 mM ethylenediaminetetraacetic acid, 300 mM NaCl and 1 mM β-mercaptoethanol as buffer. For work in darkness, appropriate infrared (940 nm ± 45 nm) visualization equipment was used.…”

“…For work in darkness, appropriate infrared (940 nm ± 45 nm) visualization equipment was used. 61 For crystallization trials, a sample of Y263F at a concentration of 10 mg/ml was irradiated with far-red light to populate the Pr state and further purified using a preparative Superdex 200 column (V t = 318 ml and V 0 = 105 ml; GE Healthcare).…”

“…All purified samples (concentration of 10 mg/ml) were handled in complete darkness using infrared visualization equipment (940 ± 45 nm). 61 To minimize photoconversion by the measuring light of the instrument, we irradiated samples with the appropriate actinic light source prior to each scan. A total of 10 scans were collected and averaged.…”

“…10,61 Samples were irradiated with light-emitting diodes and appropriate interference filters (651 nm for Y263F and 640 nm for Y263H and Y263S). Extinction coefficients were determined by measuring equal concentrations of the holoproteins in β-mercaptoethanol buffer (native conditions) and in 8 M urea at pH 2.0 (acidic denaturing conditions) based on the extinction coefficient of phycocyanin.…”