Protein kinetics: structures of intermediates and reaction mechanism from time-resolved x-ray data.
We determine the number of authentic reaction intermediates in the later stages of the photocycle of photoactive yellow protein at room temperature, their atomic structures, and a consistent set of chemical kinetic mechanisms, by analysis of a set of time-dependent difference electron density maps spanning the time range from 5 micros to 100 ms. The successful fit of exponentials to right singular vectors derived from a singular value decomposition of the difference maps demonstrates that a chemical kinetic mechanism holds and that structurally distinct intermediates exist. We identify two time-independent difference maps, from which we refine the structures of the corresponding intermediates. We thus demonstrate how structures associated with intermediate states can be extracted from the experimental, time-dependent crystallographic data. Stoichiometric and structural constraints allow the exclusion of one kinetic mechanism proposed for the photocycle but retain other plausible candidate kinetic mechanisms.
Schmidt
Marius
M
Physikdepartment E17, Technische Universität München, 85747 Garching, Germany. marius@hexa.e17.physik.tu-muenchen.de
Pahl
Reinhard
R
Srajer
Vukica
V
Anderson
Spencer
S
Ren
Zhong
Z
Ihee
Hyotcherl
H
Rajagopal
Sudarshan
S
Moffat
Keith
K
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D007700:Kinetics; D020283:Photoreceptors, Microbial / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria