Solution structure and backbone dynamics of the photoactive yellow protein.

The solution structure of photoactive yellow protein (PYP), a photosensory protein from Ectothiorhodospira halophila, has been determined by multidimensional NMR spectroscopy. The structure consists of an open, twisted, 6-stranded, antiparallel beta-sheet, which is flanked by four alpha-helices on both sides. The final set of 26 selected structures is well-defined for the regions spanning residues Phe6-Ala16, Asp24-Ala112, and Tyr118-Val125 and displays a root-mean-square deviation, versus the average, of 0.45 A for the backbone and 0.88 A for all heavy atoms. Comparison of the solution structure with an earlier published 1.4 A crystal structure (Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278-6287) reveals a similarity with a root-mean-square deviation of 1.77 A for the backbone for the well-defined regions. The most distinct difference in the backbone with the crystal structure is found near the N-terminus, for residues Asp19-Leu23, which corresponds to an alpha-helix in the crystal structure and to one of the poorest defined regions in the solution structure. To characterize the dynamic behavior of PYP in solution, we undertook a 15N relaxation study and measurements of hydrogen/deuterium exchange. Determination of order parameters through the model-free Lipari-Szabo approach enabled the identification of several regions of enhanced dynamics. The comparison of atomic displacements in the backbone traces of the ensemble structures, with mobility measurements from NMR, show that the poorly defined regions feature fast internal motions in the nanosecond to picosecond time scale.

Structural and dynamic changes of photoactive yellow protein during its photocycle in solution.

Light irradiation of photoactive yellow protein (PYP) induces a photocycle, in which red-shifted (pR) and blue-shifted (pB) intermediates have been characterized. An NMR study of the long-lived pB intermediate now reveals that it exhibits a large degree of disorder and exists as a family of multiple conformers that exchange on a millisecond time scale. This shows that the behavior of PYP in solution is different from what has been observed in the crystalline state. Furthermore, differential refolding to ground state pG is observed, whereby the central beta-sheet and parts of the helical structure are formed first and the region around the chromophore at a later stage.

Solution structure of a Lewis(x) analogue by off-resonance 1H NMR spectroscopy without use of an internal distance reference.

A recent 1H NMR method has been applied to the determination of the solution structure and internal dynamics of a synthetic mixed C/O trisaccharide related to sialyl Lewis(x). Varying the rf field offset in ROESY-type experiments enabled the measurement of longitudinal and transverse dipolar cross-relaxation rates with high accuracy. Assuming that for each proton pair the motion could be represented by a single exponential autocorrelation function, it was possible to derive geometrical parameters (r) and dynamic parameters (tau(cp)). With this assumption, 224 cross-relaxation rates have been transformed into 30 interproton distance constraints and 30 dipolar correlation times. The distance constraints have been used in a simulated-annealing procedure. This trisaccharide exhibits a structure close to the O-glycosidic analogue, but its flexibility seems highly reduced. On the basis of the determined structure and dynamics, it is shown that no conformational exchange occurs, the molecule existing in the form of a unique family in aqueous solution. In order to assess the quality of the resulting structures and to validate this new experimental procedure of distance extraction, we finally compare these solution structures to the ones obtained using three different sets of distances deduced from three choices of internal reference. It appears that this procedure allows the determination of the most precise and accurate solution.