TIME-RESOLVED SMALL-ANGLE X-RAY SCATTERING OF JUMP-RELAXATION AND STOPPED-FLOW EXPERIMENTS OF LIPIDS AND PROTEINS
M. Kriechbaum1, M. Steinhart1, P. Laggner1, Y. Hiragi2, H. Amenitsch3 and S. Bernstorff3
1Institute of Biophysics and X-Ray
Structure Research, Austrian Academy of Sciences, Graz, AUSTRIA,
2Institute for Chemical Research, Kyoto
University, Kyoto, JAPAN,
3 Sincrotrone Trieste, Trieste, ITALY.
Time-resolved jump-relaxation experiments on biological samples (lipids, proteins) are a suitable method to elucidate the kinetics and dynamics governing the structural rearrangements during a phase transition. We have applied this technique using pressure-jump, temperature-jump and stopped-flow on phase transitions of lipids and unfolding of proteins induced by chemical denaturation monitored by time-resolved small-angle X-ray scattering with millisecond time resolution on the SAX-beamline at ELETTRA, Trieste, Italy.
Barotropic phase transitions of the phospholipid DOPE induced by pressure-jumps using jump amplitudes of up to 3 kbar within 10 ms were investigated with a high-pressure X-ray cell [1,2] within a temperature region of 5-70°C and a pressure region of 1-3000 bar. Within this T and p range DOPE/water system exhibits two lamellar (gel and liquid-crystalline) and a non-lamellar hexagonal-inverted phase.
Chemical denaturation studies of the chaperonin complex
GroEl-ATP were investigated in a stopped flow apparatus. GroEL, a
protein assembled of 7 subunits, forming a heptameric hollow
cylinder, was rapidly mixed (100 s) with 6M GuHCl and the
denaturation was followed by time-sliced X-ray diffraction
patterns. Depending on the admixture of various ligands and ions
the denaturation (dissociation of the heptamer and unfolding of
the monomers) had different time constants.