Biomedical subjects

Philip A Anfinrud

Publications and source records attributed to Philip A Anfinrud.

9 recordsLinked to original sources

PubMed2006

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. After photolysis the carbon monoxide moves to a docking site, causing rearrangements in the heme-pocket residues, the coordinate changes of which can be plotted as a function of time. These include rotations of the heme-pocket phenylalanine concomitant with movement of the distal histidine toward the solvent, potentially allowing carbon monoxide movement in and out of the protein and proximal displacement of the heme iron. The degree of relaxation toward the intermediate and deoxy states was probed by analysis of the coordinate movements in the time-resolved models, revealing a non-linear progression toward the unbound state with coordinate movements that begin in the heme-pocket area and then propagate throughout the rest of the protein.

Algorithms↗

PubMed2006

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. Photodissociation of MbCO generates a nonequilibrium population of protein structures relaxing over a time range extending from picoseconds to milliseconds. This process triggers ligand migration to matrix cavities with clear-cut effects on the rate and yield of geminate rebinding. Here, we report subnanosecond time-resolved Laue diffraction data on the triple mutant YQR-Mb [Leu-29(B10)Tyr, His-64(E7)Gln, Thr-67(E10)Arg] that depict the sequence of structural events associated with heme and protein relaxation from 100 ps to 316 ns and above. The photodissociated ligand rapidly (<0.1 ns) populates the Xe-binding cavity distal to the heme. Moreover, the heme relaxation toward the deoxy configuration is heterogeneous, with a slower phase ( approximately ns) evident in these experiments. Damping of the heme response appears to result from a strain exerted by the E-helix via the CD-turn; Phe-43(CD1), in close contact with heme, opposes tilt until the strain is relieved. A comparison with crystallographic data on wild-type Mb and mutants Leu(29)Phe or Leu(29)Trp suggests that the internal structure controls the rate and amplitude of the relaxation events. A correlation between structural dynamics as unveiled by Laue crystallography and functional properties of Mb is presented.

Carbon Monoxide↗

PubMed2005

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. We identify a complex chemical mechanism characterized by five distinct structural intermediates. Structural changes at the chromophore in the early, red-shifted intermediates are transduced to the exterior of the protein in the late, blue-shifted intermediates through an initial "volume-conserving" isomerization of the chromophore and the progressive disruption of hydrogen bonds between the chromophore and its surrounding binding pocket. These results yield a comprehensive view of the PYP photocycle when seen in the light of previous biophysical studies on the system.

Bacterial Proteins↗

PubMed2005

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. In addition, we use more conventional transient absorption methods to probe the dynamics of ligand escape from this site, as well as the ultrafast dynamics of NO geminate recombination with the active binding site. The systems investigated include myoglobin, hemoglobin, and microperoxidase.

Animals↗

PubMed2004

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. From 1,700 single-molecule trajectories, we identified and structurally characterized a conformational switch that directs dissociated ligands to one of two nearby protein cavities. Subsequent ligand migration proceeds through a network of transiently interconnected internal cavities, with passage between them involving correlated protein-ligand motions. The simulations also suggest how picosecond protein motions modulate the functional dissociation of oxygen and suppress the geminate recombination of toxic carbon monoxide.

Carbon Monoxide↗

PubMed2004

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. The polarization anisotropy of bound CO is virtually the same for all protein systems investigated and is unchanging across its inhomogeneously broadened mid-IR absorption spectrum. From these results, it was concluded that the transition dipole moment of bound CO is oriented </=7 degrees from the heme plane normal. The polarized absorbance spectra of docked CO are similar for all protein systems investigated, but in stark contrast to bound CO, the polarization anisotropy is strongly correlated with vibrational frequency. The frequency-dependent anisotropy imposes severe constraints on the orientational probability distribution function of the transition dipole, which is well described as a dipole bathed in a Stark field whose out-of-plane motion is constrained by a simple double-well potential. The orientational and spatial constraints imposed on docked CO by the surrounding highly conserved amino acids serve to mediate ligand transport to and from the binding site and thereby control the rates and pathways for geminate ligand rebinding and ligand escape.

Animals↗

PubMed2004

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. The variations in the distance between pairs of chlorine atoms in adjacent CCl4 molecules are probed on the mA length scale. However, the most striking outcome of the present work is the experimental determination of temporally varying atom-atom pair distribution functions. Variations of the mean density of the solution during thermal expansion are also followed in real time. One concludes that not only time-resolved optical spectroscopy but also time-resolved x-ray diffraction can be used to monitor atomic motions in liquids.

Carbon Tetrachloride↗

PubMed2004

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. The sub-ns structural rearrangements that accompany ligand dissociation in wild-type and the mutant form differ dramatically, and lead to vastly different ligand migration dynamics. The correlated protein displacements provide a structural explanation for the kinetic differences. Our observation of functionally important protein motion on the sub-ns time scale was made possible by the 150-ps time resolution of the measurement, and demonstrates that picosecond dynamics are relevant to protein function. To visualize subtle structural changes without modeling, we developed a novel method for rendering time-resolved electron density that depicts motion as a color gradient across the atom or group of atoms that move. A sequence of these time-resolved images have been stitched together into a movie, which allows one to literally "watch" the protein as it executes its function.

Amino Acid Substitution↗

PubMed2003

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. Time-resolved mid-infrared spectroscopy of flash-photolyzed L29F MbCO revealed a short-lived CO intermediate whose 140-ps lifetime is shorter than that found in wild-type protein by a factor of 1000. The electron density maps of the protein unveil transient conformational changes far more dramatic than the structural differences between the carboxy and deoxy states and depict the correlated side-chain motion responsible for rapidly sweeping CO away from its primary docking site.

Amino Acid Substitution↗