Proteins are engaged in a myriad of tasks that are essential to life. The total number of these life-sustaining molecules is enormous. For example, drafts of the human genome contain 30,000-40,000 protein-coding genes that, through splicing, generate an even larger number of protein products. To better understand how proteins execute their designed function, much effort has gone into the determination of their three-dimensional structures. For example, over 12,000 protein structures have been deposited into the Protein Data Bank, of which nearly 3,300 are characterized as enzymes. Static structures of enzymes have proven quite enlightening; however, the side chains surrounding the active site of an enzyme are not static, passive spectators, but are active participants in the choreographed motions that mediate chemical transformation. To fully understand how an enzyme functions at the molecular level, it is crucial to know the structural changes that ensue as it executes its designed function. With this knowledge, researchers will be far better poised to rationally engineer proteins and peptides with therapeutic value. To that end, we have collaborated with an international team to develop the technique of picosecond X-ray crystallography. This technique is based on the pump-probe method, where a picosecond laser pulse (pump) triggers a reaction in a protein crystal and a delayed X-ray pulse (probe) takes a """"""""snapshot"""""""" of the protein?s structure at a time controlled by the pump-probe delay. A frame-by-frame sequence of snapshots acquired at different delay times allows us to literally watch a protein as it functions. The model system chosen to assess the capabilities of picosecond macromolecular crystallography is the carbon monoxide (CO) complex of the L29F mutant of myoglobin. This mutant, where the leucine (L) in the 29 position is replaced by phenylalanine (F), exhibits unusually rapid ligand dynamics. For example, time-resolved mid-IR spectroscopy revealed a short-lived (140 ps) intermediate whose disappearance evidently corresponds to CO hopping from a nearby docking site to other cavities within the protein. Because this time constant is comparable to the current time-resolution of X-ray crystallography, this mutant provides a stringent test of its capabilities. Thanks to numerous improvements in the X-ray and laser equipment (on the ID09B beam line at the European Synchrotron and Radiation Facility in Grenoble, France), a significant breakthrough in this venture was recently realized. In the spring of 2002, time-resolved """"""""snapshots"""""""" of this mutant?s crystal structure captured a """"""""transition state"""""""" whose highly strained side chains apparently sweep CO away from its primary docking site on the sub-ns time scale. Moreover, we believe we have also identified a site to which CO is swept. By extending the time-resolution of crystallography into a time domain readily accessible to molecular dynamics simulations, we have paved the way to explore functionally-important structure transitions both theoretically and experimentally, from which an ever more atomistic description of protein function will be achieved. Our breakthrough in picosecond crystallography was made possible, in part, by femtosecond time-resolved studies of the photophysics of heme proteins in crystals. A state-of-the-art femtosecond time-resolved uv-visible spectrometer, constructed in LCP, was used to study transient photolysis intermediates of MbCO crystals under high intensity laser excitation. At the high fluence levels required to excite a significant fraction of the protein within a crystal, the probability of a second photon being absorbed by an excited state increases as the pump pulse becomes shorter. This multi-photon absorption evidently leads to the formation of met-myoglobin, which doesn?t bind gaseous ligands. By stretching the 100 femtosecond laser pulse to a few picoseconds prior to MbCO photolysis, the protein crystal was more tolerant to repeated laser excitation, allowing multiple time points to be measured with a single protein crystal. Our ability to characterize photophysical processes in protein crystals on the ultrafast time scale has proven to be an essential complement to picosecond time-resolved X-ray crystallography.
| Aranda 4th, Roman; Levin, Elena J; Schotte, Friedrich et al. (2006) Time-dependent atomic coordinates for the dissociation of carbon monoxide from myoglobin. Acta Crystallogr D Biol Crystallogr 62:776-83 |
| Bourgeois, Dominique; Vallone, Beatrice; Arcovito, Alessandro et al. (2006) Extended subnanosecond structural dynamics of myoglobin revealed by Laue crystallography. Proc Natl Acad Sci U S A 103:4924-9 |
| Anfinrud, Philip; Schotte, Friedrich (2005) Chemistry. X-ray fingerprinting of chemical intermediates in solution. Science 309:1192-3 |
| Lim, Manho; Anfinrud, Philip A (2005) Ultrafast time-resolved IR studies of protein-ligand interactions. Methods Mol Biol 305:243-60 |
| Ihee, Hyotcherl; Rajagopal, Sudarshan; Srajer, Vukica et al. (2005) Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds. Proc Natl Acad Sci U S A 102:7145-50 |
| Plech, Anton; Wulff, Michael; Bratos, Savo et al. (2004) Visualizing chemical reactions in solution by picosecond x-ray diffraction. Phys Rev Lett 92:125505 |
| Lim, Manho; Jackson, Timothy A; Anfinrud, Philip A (2004) Orientational distribution of CO before and after photolysis of MbCO and HbCO: a determination using time-resolved polarized Mid-IR spectroscopy. J Am Chem Soc 126:7946-57 |
| Hummer, Gerhard; Schotte, Friedrich; Anfinrud, Philip A (2004) Unveiling functional protein motions with picosecond x-ray crystallography and molecular dynamics simulations. Proc Natl Acad Sci U S A 101:15330-4 |
| Anderson, Spencer; Srajer, Vukica; Pahl, Reinhard et al. (2004) Chromophore conformation and the evolution of tertiary structural changes in photoactive yellow protein. Structure 12:1039-45 |
| Bourgeois, Dominique; Vallone, Beatrice; Schotte, Friedrich et al. (2003) Complex landscape of protein structural dynamics unveiled by nanosecond Laue crystallography. Proc Natl Acad Sci U S A 100:8704-9 |
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