A Molecular Movie at 1.8 Å Resolution Displays the Photocycle of Photoactive Yellow Protein, a Eubacterial Blue-Light Receptor, from Nanoseconds to Seconds

et al. 2006

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111

Protein allostery provides mechanisms for regulation of biological function at the molecular level. We present here an investigation of global, ligand-induced allosteric transition in a protein by time-resolved x-ray diffraction. The study provides a view of structural changes in single crystals of Scapharca dimeric hemoglobin as they proceed in real time, from 5 ns to 80 s after ligand photodissociation. A tertiary intermediate structure forms rapidly (<5 ns) as the protein responds to the presence of an unliganded heme within each R-state protein subunit, with key structural changes observed in the heme groups, neighboring residues, and interface water molecules. This intermediate lays a foundation for the concerted tertiary and quaternary structural changes that occur on a microsecond time scale and are associated with the transition to a low-affinity T-state structure. Reversal of these changes shows a considerable lag as a T-like structure persists well after ligand rebinding, suggesting a slow T-to-R transition.allosteric protein transitions ͉ intersubunit communication ͉ kinetics A fundamental goal in biology is to understand how organisms respond to environmental signals. Central to this process are allosteric proteins that undergo structural transitions between alternate states in response to stimuli such as ligand binding. Although static structures of alternate states are available for a number of allosteric proteins, information about the kinetic pathway between such functionally important states is limited. Time-resolved crystallographic analysis (1-9) provides the unique opportunity to obtain direct, time-dependent structural information at high resolution on the entire protein molecule as it undergoes structural change.Much of our understanding of allosteric protein function has come from investigations into mammalian hemoglobins. Evidence of substantial structural changes accompanying ligand binding dates back to the 1930s with the observation that unliganded, high-salt, hemoglobin crystals exposed to oxygen shatter (10), foreshadowing the discovery of large quaternary changes that are linked to oxygen binding (11). Unliganded crystals of human hemoglobin grown under certain low-salt conditions can maintain their integrity upon oxygen binding; however, oxygen binding in this case is noncooperative (12). Although the ability to follow allosteric transitions in the crystalline state is limited when such large quaternary structural changes accompany cooperative ligand binding, spectroscopic investigations of hemoglobin solutions have revealed some aspects of the kinetic pathway of allosteric structural transitions (13-15). Results to date suggest a multistep pathway, with key tertiary structural transitions taking place within a few microseconds and quaternary rearrangements occurring in the range of tens of microseconds. In addition to providing structural information on high-affinity R and low-affinity T quaternary forms at the atomic level (16-18), crystallographic experiments have also succeeded...

Section: Methodsmentioning

confidence: 99%

Section: Allosteric Protein Transitions ͉ Intersubunit Communication mentioning

confidence: 99%

Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

Knapp

Pahl²,

et al. 2006

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111

“…By comparing the structure amplitudes before initiation with those after a time delay, we obtain timedependent difference electron density maps that provide a detailed picture of the structural changes as the reaction proceeds, without any trapping (13)(14)(15)(16)(17)(18). However, whether or not a simple chemical mechanism holds, several ISs are likely to be populated at all time delays; the difference electron density features associated with each intermediate overlap in each map.…”

mentioning

confidence: 99%

“…PYP is a blue light photoreceptor believed to be involved in the regulation of negative phototaxis (22) in various photosynthetic bacteria (23). Absorption of a photon of blue light triggers trans-to-cis isomerization of the chromophore of PYP, p-coumaric acid, and entry into a photocycle containing a series of at least six spectroscopically distinct states (24)(25)(26)(27)(28) (15). Here, we quantitatively determine the structures of intermediates and identify plausible kinetic mechanisms from an entire time course of difference maps spanning the later stages of the photocycle of PYP.…”

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confidence: 99%

Protein kinetics: Structures of intermediates and reaction mechanism from time-resolved x-ray data

Schmidt

Pahl

et al. 2004

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We determine the number of authentic reaction intermediates in the later stages of the photocycle of photoactive yellow protein at room temperature, their atomic structures, and a consistent set of chemical kinetic mechanisms, by analysis of a set of time-dependent difference electron density maps spanning the time range from 5 s to 100 ms. The successful fit of exponentials to right singular vectors derived from a singular value decomposition of the difference maps demonstrates that a chemical kinetic mechanism holds and that structurally distinct intermediates exist. We identify two time-independent difference maps, from which we refine the structures of the corresponding intermediates. We thus demonstrate how structures associated with intermediate states can be extracted from the experimental, time-dependent crystallographic data. Stoichiometric and structural constraints allow the exclusion of one kinetic mechanism proposed for the photocycle but retain other plausible candidate kinetic mechanisms.chemical, kinetic mechanism ͉ time-resolved crystallography T he protein structures associated with short-lived intermediate states (ISs) that form and decay along a reaction pathway must be determined if the structural basis of function is to be fully understood. Models for such states are normally determined by physical or chemical trapping techniques designed both to greatly increase the lifetime of a particular state and to trap a structurally homogeneous species (1-4). However, the trapping techniques themselves may perturb the mechanism and the observed species may still not be structurally homogeneous (5). If homogeneous structures along the reaction path could be determined directly by using unmodified protein and reactants at near-physiological temperatures, the difficulties associated with trapping could be overcome.The reaction pathway of a protein is typically described by a chemical kinetic mechanism populated by a reactant state, a set of ISs, and a product state. For such a mechanism to hold, the transit time of an individual molecule from each state to its neighboring state(s) must be short, compared with the lifetimes of the states themselves. That is, equilibration across all other degrees of freedom must be fast with respect to progress along the reaction pathway. The time scales of structural transitions in proteins range from picoseconds for side-chain relaxations (6) to several nanoseconds for protein backbone relaxations (7). Such protein-specific motions are thought to enable both the transitions between protein conformations or substates within a single state (8,9), and those between ISs. If the lifetime of an IS is short compared with the time scales of protein-specific motions, a simple chemical kinetic mechanism (10) is unlikely to hold. If the converse is true, those distinct ISs that accumulate to sufficiently high concentration will be observable. If first-order reactions are considered, the rise and fall of the concentration of each state will be describable by a sum of exponentials...

“…A laser pulse and subsequent train of synchrotron x-ray pulses represent a pump-probe experiment, in which the temporal resolution can be as short as 150 ps (25). Time-resolved crystallography with nanosecond time resolution has been successfully employed to observe the photolysis, protein relaxation, and rebinding of carbon monoxide from myoglobin (26) and the photocycle of photoactive yellow protein (27).…”

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confidence: 99%

Time-resolved crystallographic studies of light-induced structural changes in the photosynthetic reaction center

Baxter

Ponomarenko

et al. 2004

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Light-induced structural changes in the bacterial reaction center were studied by a time-resolved crystallographic experiment. Crystals of protein from Blastochloris viridis (formerly Rhodopseudomonas viridis) were reconstituted with ubiquinone and analyzed by monochromatic and Laue diffraction, in the dark and 3 ms after illuminating the crystal with a pulsed laser (630 nm, 3 mJ͞pulse, 7 ns duration). Refinement of monochromatic data shows that ubiquinone binds only in the ''proximal'' Q B binding site. No significant structural difference was observed between the light and dark datasets; in particular, no quinone motion was detected. This result may be reconciled with previous studies by postulating equilibration of the ''distal'' and ''proximal'' binding sites upon extended dark adaption, and in which movement of ubiquinone is not the conformational gate for the first electron transfer between Q A and QB.bacterial photosynthesis ͉ secondary electron transfer ͉ Laue diffraction ͉ time-resolved crystallography ͉ quinones