3 research outputs found
Homology Modeling and Molecular Dynamics Simulation Combined with X‑ray Solution Scattering Defining Protein Structures of Thromboxane and Prostacyclin Synthases
A combination of
molecular dynamics (MD) simulations and X-ray
scattering (SAXS) has emerged as the approach of choice for studying
protein structures and dynamics in solution. This approach has potential
applications for membrane proteins that neither are soluble nor form
crystals easily. We explore the water-coupled dynamic structures of
thromboxane synthase (TXAS) and prostacyclin synthase (PGIS) from
scanning HPLC–SAXS measurements combined with MD ensemble analyses.
Both proteins are heme-containing enzymes in the cytochrome P450 family,
known as prostaglandin H<sub>2</sub> (PGH<sub>2</sub>) isomerase,
with counter-functions in regulation of platelet aggregation. Currently,
the X-ray crystallographic structures of PGIS are available, but those
for TXAS are not. The use of homology modeling of the TXAS structure
with ns−μs explicit water solvation MD simulations allows
much more accurate estimation of the configuration space with loop
motion and origin of the protein behaviors in solution. In contrast
to the stability of the conserved PGIS structure in solution, the
pronounced TXAS flexibility has been revealed to have unstructured
loop regions in connection with the characteristic P450 structural
elements. The MD-derived and experimental-solution SAXS results are
in excellent agreement. The significant protein internal motions,
whole-molecule structures, and potential problems with protein folding,
crystallization, and functionality are examined
Probing Ligand Binding to Thromboxane Synthase
Various fluorescence experiments and computer simulations
were utilized to gain further understanding of thromboxane A<sub>2</sub> synthase (TXAS), which catalyzes an isomerization of prostaglandins
H<sub>2</sub> to give rise to thromboxane A<sub>2</sub> along with
a fragmentation reaction to 12-l-hydroxy-5,8,10-heptadecatrienoic
acid and malondialdehyde. In this study, 2-<i>p</i>-toluidinylnaphthalene-6-sulfonic
acid (TNS) was utilized as a probe to assess the spatial relationship
and binding dynamics of ligand–TXAS interactions by steady-state
and time-resolved fluorescence spectroscopy. The proximity between
TNS and each of the five tryptophan (Trp) residues in TXAS was examined
through the fluorescence quenching of Trp by TNS via an energy transfer
process. The fluorescence quenching of Trp by TNS was abolished in
the W65F mutant, indicating that Trp65 is the major contributor to
account for energy transfer with TNS. Furthermore, both competitive
binding experiments and the computer-simulated TXAS structure with
clotrimazole as a heme ligand strongly suggest that TXAS has a large
active site that can simultaneously accommodate TNS and clotrimazole
without mutual interaction between TNS and heme. Displacement of TNS
by Nile Red, a fluorescence dye sensitive to environmental polarity,
indicates that the TNS binding site in TXAS is likely to be hydrophobic.
The Phe cluster packing near the binding site of TNS may be involved
in facilitating the binding of multiple ligands to the large active
site of TXAS
Probing Water Environment of Trp59 in Ribonuclease T1: Insight of the Structure–Water Network Relationship
In
this study, we used the tryptophan analogue, (2,7-aza)ÂTrp, which
exhibits water catalyzed proton transfer isomerization among N(1)-H,
N(7)-H, and N(2)-H isomers, to probe the water environment of tryptophan-59
(Trp59) near the connecting loop region of ribonuclease Tl (RNase
T1) by replacing the tryptophan with (2,7-aza)ÂTrp. The resulting (2,7-aza)ÂTrp59
triple emission bands and their associated relaxation dynamics, together
with relevant data of 7-azatryptophan and molecular dynamics (MD)
simulation, lead us to propose two Trp59 containing conformers in
RNase T1, namely, the loop-close and loop-open forms. Water is rich
in the loop-open form around the proximity of (2,7-aza)ÂTrp59, which
catalyzes (2,7-aza)ÂTrp59 proton transfer in the excited state, giving
both N(1)-H and N(7)-H isomer emissions. The existence of N(2)-H isomer
in the loop-open form, supported by the MD simulation, is mainly due
to the specific hydrogen bonding between N(2)-H proton and water molecule
that bridges N(2)-H and the amide oxygen of Pro60, forming a strong
network. The loop-close form is relatively tight in space, which squeezes
water molecules out of the interface of α-helix and β2
strand, joined by the connecting loop region; accordingly, the water-scant
environment leads to the sole existence of the N(1)-H isomer emission.
MD simulation also points out that the Trp-water pairs appear to preferentially
participate in a hydrogen bond network incorporating polar amino acid
moieties on the protein surface and bulk waters, providing the structural
dynamic features of the connecting loop region in RNase T1