1 H-15 N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.
Phytochromes (Phys)3 include a large and diverse superfamily of sensory photoreceptors that use a bilin (or linear tetrapyrrole) chromophore for light detection (1-3). These chromoproteins were first discovered in higher plants based on their control of many agriculturally relevant processes and have since been found by sequence searches in lower plants, algae, and numerous proteobacteria, cyanobacteria, and fungi. For photoautotrophic organisms, Phys are particularly important for optimizing photosynthetic potential where they measure the fluence rate, duration, and the direction of light and help detect shading by competitors (1, 4).The signature feature of canonical Phys is their ability to photoconvert between two stable states, a red light-absorbing Pr form that is typically the parent state of the photoreceptor, and a far-red light-absorbing Pfr form that is often biologically active (1-3) (for examples of exceptions see Refs. 5-7). This activity is directed by a series of structural domains that play specific roles in photoperception. A large N-terminal region encompasses the chromophore-binding domain (CBD); it includes a proximal Per/Arndt/Sim (PAS) domain immediately followed by a cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domain. The CBD autocatalytically attaches the bilin via a thioether linkage and together with the chromophore generates the unique photochromic absorption properties of Phys.For proteobacterial (BphP) and fungal Phys, biliverdin (BV) is used as the chromophore (8). BV is synthesized by oxidative cleavage of heme by a heme oxygenase (HO) and then attached to the apoprotein through its A-pyrrole ring vinyl side chain to a positionally conserved cysteine upstream of the PAS domain (9, 10). For cyanobacterial (Cph) and plant Phys, phycocyanobilin (PCB) and phytochromobilin (P⌽B) are used as the chromophore, respectively (2,11,12). PCB/P⌽B are synthesized by enzymatic reduction of BV using a BV reductase (BVR) and then attached through their A-ring ethylidene side chains to a