Time resolved crystallography

23 Time-Resolved Crystallography. Time-resolved crystallography (TC) uses an intense synchrotron X-ray source and Laue data collection techniques to greatly reduce crystallographic exposure times. Normal time resolution for X-ray 

The results discussed here indicate that time-resolved crystallography should continue to evolve and that its use will enhance understanding of more complex enzymatic systems in the future. 

X-ray crystallography is the method of choice for determination of structures of large macromolecules such as proteins. Nowadays, roughly 48,000 x-ray structures are stored in the Protein Data Bank (http //www.rcsb.org Berman et al. 2000). X-ray crystallography is traditionally a static method, i.e., without time resolution. In order to follow the kinetics and to determine the structure of the transiently occupied intermediate states of proteins, time-resolved crystallography has to be used (Moffat 1989). The time resolution, has to be as good as for any other method employed to follow reaction kinetics. This implies that x-ray data must be collected as fast as possible. 

In third-generation synchrotrons, the x-rays are generated in intense flashes of 100 picoseconds (ps) duration. If, dnring this time, an entire diffraction pattern is recorded, the time resolution equals 100 ps (Szebenyi et al. 1992 Srajer et al. 1996, Schotte et al. 2003). However, the traditional monochromatic oscillation diffraction method cannot be used since there is no way to rotate the crystal during this 100 ps timeframe to collect the integrated intensity of a Bragg reflection. Still exposures, therefore, have to be used. 

Although other methods could be used to determine the integrated intensity, the Lane method (Amoros et al. 1975 Bartunik et al. 1992 Ren et al. 1999) has been the method of choice so far. In this method, the crystal is subjected to a spectrum of x-ray radiation. Each reflection accepts a small fraction of this bandwidth, which covers the entire reflection range of that particular reflection. Hence, the integrated 

Using time-resolved crystallographic experiments, molecular structure is eventually linked to kinetics in an elegant fashion. The experiments are of the pump-probe type. Preferentially, the reaction is initiated by an intense laser flash impinging on the crystal and the structure is probed a time delay. At, later by the x-ray pulse. Time-dependent data sets need to be measured at increasing time delays to probe the entire reaction. A time series of structure factor amplitudes, IF, , is obtained, where the measured amplitudes correspond to a vectorial sum of structure factors of all intermediate states, with time-dependent fractional occupancies of these states as coefficients in the summation. Difference electron densities are typically obtained from the time series of structure factor amplitudes using the difference Fourier approximation (Henderson and Moffatt 1971). Difference maps are correct representations of the electron density distribution. The linear relation to concentration of states is restored in these maps. To calculate difference maps, a data set is also collected in the dark as a reference. Structure factor amplitudes from the dark data set, IFqI, are subtracted from those of the time-dependent data sets, IF,I, to get difference structure factor amplitudes, AF,. Using phases from the known, precise reference model (i.e., the structure in the absence of the photoreaction, which may be determined from 

FIGURE 1.5 The heme pocket of myoglobin. B-site CO found here also at cryogenic temperatures. Xel...Xe4 sites identified in xenon binding experiments. 

---

V. Srajer, T. Teng, T. Ursby, C. Pradervand, Z. Ren, S. Adachi, W. Schildkamp, D. Bourgeois, M. WuRf, and K. Moffat, Photolysis of the carbon monoxide complex of myoglobin nanosecond time-resolved crystallography. Science 274, 1726-1729 (1996). 

Genick, U. K., G. E. Borgstahl, K. Ng et al. (1997). Structure of aprotein photocycle intermediate by millisecond time-resolved crystallography. Science 275 1471-1475. 

Misumi, S. Recognitory Coloration of Cations with Chromoacerands. 165, 163-192 (1993). Moffat, J. K., Helliwell, J. The Laue Method and its Use in Time-Resolved Crystallography. 151, 61-74 (1989). 

Nanosecond time-resolved crystallography of MbCO has been discussed in Section 3.7.2.3 of Chapter 3.46 After firing a 10-ns burst of laser light to break the CO-Fe bond, these researchers produced a diffraction image of the crystal through application of a 150-ps X-ray pulse. They are able to show release of the CO molecule, displacement of the Fe ion toward the proximal histidine, and recombination of the dissociated CO by about 100 ps. Essentially their results compare well with other spectroscopic studies of HbCO, MbCO and their models. 

Through this modification, they hoped to capture a later conformational intermediate poised to form an inline transition state. The technique used was that of time-resolved crystallography, one that has successfully captured protein intermediates (see Section 3.7.2.3). The data determining the crystal structure has been deposited in the Protein Databank as PDB 379D. 

Moffat JK, Helliwell J (1989) The Laue Method and its Use in Time-Resolved Crystallography. 151 61-74... 

Effective ligand rebinding from the Xel site is only observed when the temperature has risen above a characteristic temperature 180 K (Figure 1.6), which is the temperature where protein dynamics sets in. However, the time-related information is lost in experiments at cryogenic temperatures. Time-resolved crystallography was applied to restore the time scale and observe undisturbed relaxations. 

Ihee, H., Rajagopal, S., Srajer, V., Pahl, R., Schmidt, M., Schotte, R, Anfinrud, P. A., Wulff, M., and Moffat, K. 2005. Visualizing chromophore isomerization in photoactive yellow protein from nanoseconds to seconds by time-resolved crystallography. Pmc. Natl. Acad. Set, USA 102 7145-50. 

Schmidt, M. 2008. Structure based kinetics by time-resolved crystallography. In Ultrashort laser pulses in biology and medicine. M. Braun, P. Gilch, W. Zinth, Eds. New York Springer Heidelberg. 

Srajer, V., Teng, T. Y, Ursby, T., Pradervand, C., Ren,Z., Adachi, S., Schildkamp, W., Bourgeois, D., Wulff, M., and Moffatt, K. 1996. Photolysis of the carbon monoxide complex of myoglobin Nanosecond time-resolved crystallography. Science 274 1726-29. 

Moffat K (1998) Time-resolved crystallography. Acta Cryst A 54 833-841... 

Cole JM, Raithby PR, Wulff M, Schotte F, Plech A, Teat SJ, Bushnell-Wye G (2002) Nanosecond time-resolved crystallography of photo-induced species case smdy and instrument development for high-resolution excited-state single-crystal structure determination. Faraday Discuss 122 119-129... 

---