Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds

Ihee, Hyotcherl; Rajagopal, Sudarshan; Šrajer, V.; Pahl, R.; Anderson, Spencer; Schmidt, Marius; Schotte, Friedrich; Anfinrud, Philip; Wulff, Michaël; Moffat, Keith

doi:10.1073/pnas.0409035102

Cited by 259 publications

(403 citation statements)

References 46 publications

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“…3, curved arrows). This unusual structure has not been trapped in cryotrapping experiments (18)(19)(20), and its lifetime is too short to be captured in prior nanosecond time-resolved Laue crystallography experiments (21)(22)(23). The driving force for generating this intermediate is the trans-to-cis photoisomerization of pCA, during which the distance between the Cys69 sulfur and the terminal phenolate oxygen atom (Fig.…”

Section: Resultsmentioning

confidence: 99%

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

Schotte

Cho

Kaila

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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179

244

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To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90°out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/ relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics.time-resolved X-ray diffraction | photoreceptor | light sensor

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Section: Resultsmentioning

confidence: 99%

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

Schotte

Cho

Kaila

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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179

244

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“…X-rays are scattered by all the atoms included in the probed sample, both solute and solvent atoms. The global scattering signal from a solution can then be decomposed into three contributions [26,[49][50][51][52], schematically indicated in figure 3b.…”

Section: Time-resolved X-ray Scattering: Theoretical Backgroundmentioning

confidence: 99%

“…Their sensitivity is limited to valence electrons and vibrational features, respectively. On the other hand, direct insights into photo-induced structural dynamics can be achieved using ultrashort X-ray [25][26][27][28] or electron [29][30][31][32] probe pulses, which are nowadays available at synchrotron facilities and high-energy electron sources, respectively. Using this approach, it is possible to obtain a molecular movie of a given photoprocess, i.e.…”

Section: Introductionmentioning

confidence: 99%

Synchrotron ultrafast techniques for photoactive transition metal complexes

Borfecchia

Garino

Salassa

et al. 2013

Phil. Trans. R. Soc. A.

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In the last decade, the use of time-resolved X-ray techniques has revealed the structure of lightgenerated transient species for a wide range of samples, from small organic molecules to proteins. Time resolutions of the order of 100 ps are typically reached, allowing one to monitor thermally equilibrated excited states and capture their structure as a function of time. This review aims at providing a general overview of the application of timeresolved X-ray solution scattering (TR-XSS) and time-resolved X-ray absorption spectroscopy (TR-XAS), the two techniques prevalently employed in the investigation of light-triggered structural changes of transition metal complexes. In particular, we herein describe the fundamental physical principles for static XSS and XAS and illustrate the theory of timeresolved XSS and XAS together with data acquisition and analysis strategies. Selected pioneering examples of photoactive transition metal complexes studied by TR-XSS and TR-XAS are discussed in depth.

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“…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)(2)(3)(4)(5)(6)(7)(8)(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.…”

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,

Šrajer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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111

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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...

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Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds

Abstract: intermediates ͉ mechanism ͉ signal transduction ͉ time-resolved crystallography ͉ singular value decomposition

Cited by 259 publications

References 46 publications

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

Synchrotron ultrafast techniques for photoactive transition metal complexes

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

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