From Milliseconds to Minutes: Melittin Self-Assembly from Concerted Non-Equilibrium Pressure-Jump and Equilibrium Relaxation Nuclear Magnetic Resonance.

Non-equilibrium kinetics techniques like pressure-jump nuclear magnetic resonance (NMR) are powerful in tracking changes in oligomeric populations and are not limited by relaxation rates for the time scales of exchange that can be probed. However, these techniques are less sensitive to minor, transient populations than are Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments. We integrated non-equilibrium pressure-jump and equilibrium CPMG relaxation dispersion data to fully map the kinetic landscape of melittin tetramerization.

Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-ray Scattering.

The capacity to respond to environmental changes is crucial to an organism's survival. Halorhodospira halophila is a photosynthetic bacterium that swims away from blue light, presumably in an effort to evade photons energetic enough to be genetically harmful. The protein responsible for this response is believed to be photoactive yellow protein (PYP), whose chromophore photoisomerizes from trans to cis in the presence of blue light.

Real-time tracking of CO migration and binding in the α and β subunits of human hemoglobin via 150-ps time-resolved Laue crystallography.

We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2 Å-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both α and β subunits from 100 ps to 10 μs. The proximity of the B site in the β subunit is about 0.

Probing anisotropic structure changes in proteins with picosecond time-resolved small-angle X-ray scattering.

We have exploited the principle of photoselection and the method of time-resolved small-angle X-ray scattering (SAXS) to investigate protein size and shape changes following photoactivation of photoactive yellow protein (PYP) in solution with ∼150 ps time resolution. This study partially overcomes the orientational average intrinsic to solution scattering methods and provides structural information at a higher level of detail. Photoactivation of the p-coumaric acid (pCA) chromophore in PYP produces a highly contorted, short-lived, red-shifted intermediate (pR0), and triggers prompt, protein compaction of approximately 0.

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

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.

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering.

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique.

Time-dependent atomic coordinates for the dissociation of carbon monoxide from myoglobin.

Picosecond time-resolved crystallography was used to follow the dissociation of carbon monoxide from the heme pocket of a mutant sperm whale myoglobin and the resultant conformational changes. Electron-density maps have previously been created at various time points and used to describe amino-acid side-chain and carbon monoxide movements. In this work, difference refinement was employed to generate atomic coordinates at each time point in order to create a more explicit quantitative representation of the photo-dissociation process.

Extended subnanosecond structural dynamics of myoglobin revealed by Laue crystallography.

Work carried out over the last 30 years unveiled the role of structural dynamics in controlling protein function. Cavity networks modulate structural dynamics trajectories and are functionally relevant; in globins they have been assigned a role in ligand migration and docking. These findings raised renewed interest for time-resolved structural investigations of myoglobin (Mb), a simple heme protein displaying a photosensitive iron-ligand bond.

Ultrafast time-resolved IR studies of protein-ligand interactions.

Time-resolved mid-IR spectroscopy combines molecular sensitivity with ultrafast capability to incisively probe protein-ligand interactions in model heme proteins. Highly conserved residues near the heme binding site fashion a ligand-docking site that mediates the transport of ligands to and from the binding site. We employ polarization anisotropy measurements to probe the orientation and orientational distribution of CO when bound to and docked near the active binding site, as well as the dynamics of ligand trapping in the primary docking site.

Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds.

Determining 3D intermediate structures during the biological action of proteins in real time under ambient conditions is essential for understanding how proteins function. Here we use time-resolved Laue crystallography to extract short-lived intermediate structures and thereby unveil signal transduction in the blue light photoreceptor photoactive yellow protein (PYP) from Halorhodospira halophila. By analyzing a comprehensive set of Laue data during the PYP photocycle (forty-seven time points from one nanosecond to one second), we track all atoms in PYP during its photocycle and directly observe how absorption of a blue light photon by its p-coumaric acid chromophore triggers a reversible photocycle.

Unveiling functional protein motions with picosecond x-ray crystallography and molecular dynamics simulations.

A joint analysis of all-atom molecular dynamics (MD) calculations and picosecond time-resolved x-ray structures was performed to gain single-molecule insights into mechanisms of protein function. Ensemble-averaged MD simulations of the L29F mutant of myoglobin after ligand dissociation reproduce the direction, amplitude, and time scales of crystallographically determined structural changes. This close agreement with experiments at comparable resolution in space and time validates the individual MD trajectories.

Picosecond time-resolved X-ray crystallography: probing protein function in real time.

A detailed mechanistic understanding of how a protein functions requires knowledge not only of its static structure, but also how its conformation evolves as it executes its function. The recent development of picosecond time-resolved X-ray crystallography has allowed us to visualize in real time and with atomic detail the conformational evolution of a protein. Here, we report the photolysis-induced structural evolution of wild-type and L29F myoglobin over times ranging from 100 ps to 3 micros.

Orientational distribution of CO before and after photolysis of MbCO and HbCO: a determination using time-resolved polarized Mid-IR spectroscopy.

The technique of time-resolved polarized mid-IR spectroscopy was used to probe the orientational distribution of carbon monoxide (CO) bound to and docked within horse myoglobin, sperm whale myoglobin, and human hemoglobin A in neutral pH solution at 283 K. An accurate determination of the orientation required that the experimentally measured polarization anisotropy be corrected for the effects of fractional photolysis in an optically thick sample. The experimental method measures the direction of the transition dipole, which is parallel to the CO bond axis when docked and nearly parallel when bound to the heme.

Visualizing chemical reactions in solution by picosecond x-ray diffraction.

We present a time-resolved x-ray diffraction study to monitor the recombination of laser-dissociated iodine molecules dissolved in CCl4. The change in structure of iodine is followed during the whole recombination process. The deexcitation of solute molecules produces a heating of the solvent and induces tiny changes in its structure.

Watching a protein as it functions with 150-ps time-resolved x-ray crystallography.

We report picosecond time-resolved x-ray diffraction from the myoglobin (Mb) mutant in which Leu29 is replaced by Phe (L29Fmutant). The frame-by-frame structural evolution, resolved to 1.8 angstroms, allows one to literally "watch" the protein as it executes its function.