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Phytochromes sense red/far-red light and control many biological processes in plants, fungi, and bacteria. Although crystal structures of dark and light adapted states have been determined, the molecular mechanisms underlying photoactivation remains elusive. Here we demonstrate that the conserved tongue region of the PHY domain of a 57kDa photosensory module of Deinococcus radiodurans phytochrome, changes from a structurally heterogeneous dark state to an ordered light activated state. The results were obtained in solution by utilizing a laser-triggered activation approach detected on the atomic level with high-resolution protein NMR spectroscopy. The data suggest that photosignaling of phytochromes relies on careful modulation of structural heterogeneity of the PHY tongue. peerReviewed

Time-resolved wide-angle x-ray scattering (TR-WAXS) is an emerging biophysical method which probes protein conformational changes with time. Here we present a comparative TR-WAXS study of native green-absorbing proteorhodopsin (pR) from SAR86 and a halogenated derivative for which the retinal chromophore has been replaced with 13-desmethyl-13-iodoretinal (13-I-pR). Transient absorption spectroscopy differences show that the 13-I-pR photocycle is both accelerated and displays more complex kinetics than native pR. TR-WAXS difference data also reveal that protein structural changes rise and decay an order-of-magnitude more rapidly for 13-I-pR than native pR. Despite these differences, the amplitude and nature of the observed helical motions are not significantly affected by the substitution of the retinal's C-20 methyl group with an iodine atom. Molecular dynamics simulations indicate that a significant increase in free energy is associated with the 13-cis conformation of 13-I-pR, consistent with our observation that the transient 13-I-pR conformational state is reached more rapidly. We conclude that although the conformational trajectory is accelerated, the major transient conformation of pR is unaffected by the substitution of an iodinated retinal chromophore.

The mammalian EH-domain containing protein 2 (EHD2) is an ATPase that controls the dynamic association of caveolae with the cell surface. For this, at least three stages are identified: membrane binding, oligomerization in ring-like structures, and detachment of EHD2. It is still unclear how ATP is used in this process and if the protein undergoes conformational changes.We show that ATP binding is necessary for the protein to insert into the lipid layer and to form oligomers. Using infrared reflection absorption spectroscopy, we detect a major conformational change of the helical domains upon the membrane binding of EHD2. Membrane binding in vivo and in vitro is further influenced by inhibitory intramolecular regions in EHD2 that reorient following membrane association to allow for protein oligomerization. ATP is consumed during detachment of EHD2 from the membrane. In addition, we find that stable association of EHD2 in oligomers at the lipid membrane, but not ATP hydrolysis is required to stabilise caveolae at the cell surface.Our findings clarify the role of ATP in the process of membrane reshaping by EHD2 and describes a regulatory structural mechanism that facilitates membrane-driven oligomerization of EHD2 and stabilisation of caveolae. En route, we pioneer the use of infrared spectroscopy to gain structural and unique orientational information on membrane associated proteins.