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Project ID: HPRI-CT-1999-50004
Finanziato nell'ambito di: FP5-HUMAN POTENTIAL
Paese: France

Liquid/protein crystallography at few 10ps

The feasibility of protein and liquid crystallography at 25ps time scale has been demonstrated at the ESRF beam line. This represents a major step forward as these application experiments shows that the time resolution for the analysis of complex reactions can be boosted by a factor of 4 compared to the duration of the synchrotron x-ray pulse (100ps).

Protein crystallography: Laue diffraction studies of ligand transport in heme proteins such as the myoglobin complex with CO and O2 and similar hemoglobin complexes have been done. For that purpose, we have traditionally used the synchrotron in 1-bunch mode. The reason for that is the high intensity per bunch that this mode offers, typically 2-3 times more photons per pulse which translates into 2-3 times shorter data collection time ANF 2-3 times fewer laser excitations. The latter is important to reduce radiation damage, in particular dehydration caused by heating. The single bunch ode has now been replaced by 4-bunch mode at half the bunch current. On the up side however, we get shorter X-ray pulses, a better focus and a longer lifetime, all in all a better X-ray beam. The x-ray pulse length in the 4-bunch mode is 100-140ps and we have used these pulses in time-slicing experiments. This technique has been described in the Result related to ultrafast x-ray beam lines at synchrotrons. The results are encouraging and we have a least seen the first images with 50ps time resolution that shows the detachment of CO in the mutant L29F of MbCO. The results are shown below. The green pockets correspond to a change in charge density of 0.23e/ Angstroms³ and the purple to - 0.23 e/Angstroms³. The level of photolysis is 25% and the Fourier map is extrapolated to 100 %. The quality of this map and its time resolution of about 50ps, lead us to believe that 25ps time resolution should become possible very soon at the expense of signal to noise.

We can say that the time slicing on proteins has been slowed down due to the low levels of photolysis that could be achieved with the 100fs laser pulses that were used initially. On the basis of these experiments, pulse stretching combined with high-pulse energy from the TOPAS laser will undoubtedly make it possible to reach at least 25ps time resolution in one or two years.

Liquid crystallography: The time slicing technique has also been used to achieve few 10ps time resolution in the study chemical reactions in liquid, and more precisely the structure of the excited states of small molecules such as I2, Br2, HgI2, CH2I2 and C2H4I2 in various solvents. These experiments can, by contrast to protein crystallography, take the full 1000Hz beam from the laser and the X-ray chopper, which makes the data collection fast and precise.

The experiments have shown that not only does X-ray diffraction probe the excited chromophores; it also probes the environment, which can be divided into the first solvation shell (cage) and beyond (bulk liquid). The last part is responsible a long-range oscillations in the Fourier maps of the electron density, which can interpreted in terms of temperature and pressure during the recombination. We have worked out a fairly complete theoretical picture of x-ray diffraction from molecules in action.

Measurements of the change in the radial electron density for I2 in methanol have been obtained. The data were taken with 100ps X-ray pulses and the time delays scanned though the X-ray pulse in steps of 25ps. The signal increases as the laser position moves from -100ps towards 0ps. The maximum amplitude is attained at 50ps where the excited state is recorded with the intensity from the full bunch. The interesting thing is that the shape of the curves changes slightly in going from -50ps to 0ps. At these early time-delays, the excited molecules have not yet thermalised, conditions that are otherwise difficult to attain.

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Risultato in breve


Michael WULFF, (Head of Material Science Group)
Tel.: +33-4-76882379
Fax: +33-4-76882542