Enzymatic reactions exhibit remarkable selectivity and efficiency, the likes of which are rarely achieved in bench-top chemical reactions. While it is clear that the biochemical prowess of an enzyme arises from the highly ordered structure of its native-folded protein, the detailed mechanism by which it functions has proven elusive. This is because proteins are not simply static macromolecules that host an active site, as depicted by their crystal structure; rather, they are dynamic molecules whose choreographed motions can gate the transport of substrate to and from the active site and can modulate over time the activity of that site. To develop a mechanistic understanding of how proteins function, it is essential to study this choreography of life at the atomic level with ps time resolution. ? ? This choreography of life is being investigated by time-resolved techniques that are based on the pump-probe method. The pump is usually a laser pulse that triggers the process we wish to investigate at a well-defined instant of time. The duration of the laser pulse can be as short as a few tens of femtoseconds, which allows us to access the chemical time scale for molecular motion. After photoexcitation, a time-delayed probe pulse is directed through the pump-illuminated volume to interrogate the system. When the probe pulse is derived from an X-ray source, we can probe the protein structure via Laue diffraction or via Wide-Angle-X-ray-Scattering (WAXS). When the probe pulse is generated in the uv-vis or mid-IR region, we can interrogate the system spectroscopically. The time resolution of the pump-probe method is limited only by the duration of the pump and probe pulses and the timing jitter between them. Each pump-probe measurement produces a time-resolved snapshot of the protein. By stitching together a series of snapshots, we can create a movie that, in the case of time-resolved Laue diffraction, provides a near-atomic view of the correlated structure changes triggered by the pump pulse. We can literally watch a protein as it functions!? ? Time-resolved Laue studies are performed using protein crystals. The intermolecular forces that maintain crystalline order give rise to high resolution diffraction spots, but those same forces constrain large amplitude conformational motion. This loss of conformational flexibility may perturb, and may even inhibit the function of a protein. Nonetheless, this method stands alone in its ability to acquire near-atomic structural information on ultrafast time scales. X-rays can also extract structural information from molecules in solution where the full range of conformational motion is permitted. Because there is no long range order in protein solutions, X-ray scattering from the protein is diffuse, but contains diffraction rings that provide a fingerprint that can be correlated with the protein structure. Though the information provided by time-resolved WAXS is not at atomic resolution, models of the protein structure can be compared against the measured scattering spectrum, and time-dependent changes of the WAXS fingerprint can be used to assess putative models that describe the reaction pathway. Our efforts to develop time-resolved X-ray methods to study protein function are summarized in separate annual reports. Since these studies all use light as a trigger, pursuing these studies requires detailed knowledge of the photophysics of proteins. Moreover, we need to be able to assess the dynamics for the time-ordered sequence of events that follow the laser trigger. Finally, we need to be able to make these measurements in protein solutions as well as crystals. Our time-resolved optical studies are the focus of this annual report.? ? We have developed an ultrafast time-resolved microfocusing spectrophotometer that is capable of probing protein dynamics in solution and in crystals. The probe pulses used in this home-built spectrometer are generated by an Optical Parametric Amplifier (OPA) whose signal output is used to produce a broadband single filament white-light continuum through nonlinear interactions with a transparent dielectric medium. The stability of this continuum depends critically on the characteristics of the laser pulse used to drive the OPA, which in turn depends critically on the stability of the room temperature in our laser lab. After reengineering the temperature control system in our laser lab, we have achieved a temperature stability of +/- 0.1 degrees C. The long-term stability of the continuum probe facilitates extensive signal averaging, and the signal-to-noise ratio of spectra now emerging from this laboratory is without precedent. We have tuned up the spectrometer via studies of ligand dynamics in myoglobin and hemoglobin and their mutants. To probe the ligand dynamics from the nanosecond to the millisecond regime, we have used a tunable nanosecond Optical Parametric Oscillator (OPO) as the pump source. The white light continuum spans from 375 to 1000 nm, thereby providing access to the Soret band, the Q-band, and the near-IR bands, including band III. Because we pursue these studies under similar conditions used in time-resolved X-ray studies, the protein concentration is too high to track the Soret band. Consequently, these studies have focused on the Q-band and band III. Our spectrometer records a complete absorption spectrum with a single 100-fs duration probe pulse. Time-resolved spectra are generally acquired on a logarithmic sequence of pump-probe time delays, and their evolution informs us about the ligation state of the heme as well as the tertiary and quaternary state of the protein.? ? To extract the maximum amount of information contained within our time-resolved spectra, we have developed a novel approach for analyzing our data. We recover via nonlinear least-squares methods the rate coefficients that describe the population dynamics of all states invoked by our model; at the same time, we extract via linear least-squares methods the time-independent basis spectra for those states. Because we solve the population dynamics problem by integrating differential equations on a logarithmic timescale, we are able to account for the saturating effects of high fluence pump pulses used in time-resolved X-ray studies. We have recently focused our attention on the dynamics associated with the allosteric R to T quaternary structure transition of hemoglobin, which we have been studying both with time-resolved WAXS and optical spectroscopy. The signal-to-noise ratio of the optical spectra is far superior to that attained with WAXS, so we use those data to define many of the rate coefficients associated with the protein and ligand dynamics. The WAXS spectra are very sensitive to the quaternary structure spectra of the hemoglobin, so the time-resolved WAXS spectra are used to characterize the dynamics associated with the R to T quaternary structure transition. The timescale for this allosteric process was found to be considerably faster than that reported in the literature. Through a combination of time-resolved optical and X-ray studies, were now able to investigate allostery in proteins at an unprecedented level of detail.
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| 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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