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Feng Zhou and Paul Bash*
Center of Mechanistic Biology and Biotechnology
Argonne National Laboratory
*current address: Northwestern University
Keith Moffat
Department of Biochemistry and Molecular Biology
University of Chicago
Alexander MacKerrell
School of Pharmacy
University of Maryland, Baltimore
Fig. 1: Ribbon diagram of the 1.4 A photoactive yellow protein structure
Even though static structures are now available for hundreds of proteins molecules, it remains a mystery of nature how proteins carry out their functions through dynamic motions and conformational changes. Many proteins, such as enzymes and gene regulatory proteins, have at least two conformational states in which the protein exists, and the transition of protein conformations between these states is tightly coupled to the protein function. Unfortunately, conventional structure determination techniques only provide a time-averaged picture of the protein, and are unsuitable to study protein conformational changes occuring on time scales anywhere from picoseconds to seconds. The time-resolved X-ray diffraction techniques, utilizing high-intensity X-ray sources available from synchrotrons such as the Argonne Advanced Photon Source, in conjunction with computational modeling techniques, however, provide an unique new opportunity to study protein conformational changes and dynamics at nanosecond to millisecond time scale. We are currently investigating using computational modeling techniques the light-induced reaction of Photoactive Yellow Protein (PYP), a protein involved in signal transduction in Eubacteria, concurrent with time-resolved Laue diffraction experiments which will be carried out for this protein at Argonne APS.
We are carrying out molecular dynamics simulations of the light-induced and subsequent conformational dynamics for PYP based on physical/chemical principles. The simulated results will, for the first time, be directly comparable to protein conformational changes measured experimentally on the same time scale.
We have first developed an empirical parameter set for the thiol ester linked p-coumaric acid chromophore, the first such chromophore found in a protein, for PYP simulations. The parameters were developed using a systematic CHARMM procedure, which fits empirical parameters using observed and ab initio molecular geometry, interaction, dipole moment, normal mode frequency as well as torsional barriers which are important for protein dynamics. Quantitative agreement was achieved for calculated and observed molecular Raman frequencies.
Molecular dynamics (MD) simulations were carried out for PYP ground state at three possible protonation states. The simulated structures had very good overall agreement with the 1.4 A x-ray structure, with a 0.69 A r.m.s. deviation for protein main chain atoms. The simulations suggest that the chromophore in PYP is partially negatively charged, in agreement with previous experiments, however it is also found that the negative charge is distributed on the nearby Tyr-42 residue part of the time, implying very close pKa values of these two groups which can be further examined using quantitative simulation techniques.
Argonne's 128-processor IBM SP parallel machine was heavily used for the parameterization and MD simulation of PYP. In order to determine the molecular properties of PYP chromophore, particularly, to determine the torsional energy surfaces around essential dihedrals, numerous 6-31 G(d), 6-31+G(d) and 6-31 G(D)/SCRF calculations were carried out using the SP nodes concurrently. The molecular dynamics simulations required running of 16 nodes in parallel for one CHARMM job, currently we are running 3 jobs simultaniously. The speed-up we gain using 16 nodes on the SP parallel computer is more than 10 times compared to using single node. We are able to produce 120 ps simulation in a working night, a nanosecond simulation could be achieved in one week, enabling us to finally compare our simulations with the experiments carried out on multi-nanosecond time frames.
Additional simulations will be carried out to predict the structure of the photo-bleached state of PYP, which ocuurs in 100 microseconds after the chromophore isomerization, and to compare with X-ray structure of the same state recently under study.
Parameterization of the excited state potential surface is under way in collaboration with Dr. Charles Martin, University of Illinois at Urbana-Champaign. The initial photoreaction and protein dynamics, occuring on nanoseconds time scale, will be studied using MD simulation and free energy perturbation. The experimental studies of the PYP dynamics at nanosecond time scale will be carried out by Prof. Keith Moffat's group using Argonne APS.
Further studies on protein protonation state, spectroscopy and recovering dynamics are also planned.
Paul Bash
Dept. of Molecular Pharmacology & Biological Chemistry
Northwestern University Medical School
Ward Bldg. Rm. 7-246
Chicago, IL 60611-3008
pabash@nwu.edu
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