Solution Structural Ensembles
of Substrate-Free Cytochrome
P450<sub>cam</sub>
Eliana
K. Asciutto
Matthew J. Young
Jeffry Madura
Susan Sondej Pochapsky
Thomas C. Pochapsky
10.1021/bi300007r.s001
https://acs.figshare.com/articles/dataset/Solution_Structural_Ensembles_of_Substrate_Free_Cytochrome_P450_sub_cam_sub_/2528185
Removal of substrate (+)-camphor from the active site
of cytochrome
P450<sub>cam</sub> (CYP101A1) results in nuclear magnetic resonance-detected
perturbations in multiple regions of the enzyme. The <sup>1</sup>H–<sup>15</sup>N correlation map of substrate-free diamagnetic Fe(II) CO-bound
CYP101A permits these perturbations to be mapped onto the solution
structure of the enzyme. Residual dipolar couplings (RDCs) were measured
for <sup>15</sup>N–<sup>1</sup>H amide pairs in two independent
alignment media for the substrate-free enzyme and used as restraints
in solvated molecular dynamics (MD) simulations to generate an ensemble
of best-fit structures of the substrate-free enzyme in solution. Nuclear
magnetic resonance-detected chemical shift perturbations reflect changes
in the electronic environment of the NH pairs, such as hydrogen bonding
and ring current shifts, and are observed for residues in the active
site as well as in hinge regions between secondary structural features.
RDCs provide information about relative orientations of secondary
structures, and RDC-restrained MD simulations indicate that portions
of a β-rich region adjacent to the active site shift so as to
partially occupy the vacancy left by removal of the substrate. The
accessible volume of the active site is reduced in the substrate-free
enzyme relative to the substrate-bound structure calculated using
the same methods. Both symmetric and asymmetric broadening of multiple
resonances observed upon substrate removal as well as localized increased
errors in RDC fits suggest that an ensemble of enzyme conformations
are present in the substrate-free form.
2016-02-21 13:43:09
Residual dipolar couplings
cytochrome P 450cam
NH pairs
substrate removal
CYP 101A results
solution Structural Ensembles
MD
region
perturbation
RDC
site shift
enzyme conformations
alignment media
solution structure