2 research outputs found
Direct Hydride Shift Mechanism and Stereoselectivity of P450<sub>nor</sub> Confirmed by QM/MM Calculations
Nitric oxide reductase (P450<sub>nor</sub>) found in Fusarium oxysporum catalyzes the reduction of nitric oxide to N<sub>2</sub>O in a multistep process. The reducing agent, NADH, is bound in the distal pocket of the enzyme, and direct hydride transfer occurs from NADH to the nitric oxide bound heme enzyme, forming intermediate <i>I.</i> Here we studied the possibility of hydride transfer from NADH to both the nitrogen and oxygen of the heme-bound nitric oxide, using quantum chemical and combined quantum mechanics/molecular mechanics (QM/MM) calculations, on two different protein models, representing both possible stereochemistries, a <i>syn-</i> and an <i>anti-</i>NADH arrangement. All calculations clearly favor hydride transfer to the nitrogen of nitric oxide, and the QM-only barrier and kinetic isotope effects are good agreement with the experimental values of intermediate <i>I</i> formation. We obtained higher barriers in the QM/MM calculations for both pathways, but hydride transfer to the nitrogen of nitric oxide is still clearly favored. The barriers obtained for the <i>syn</i>, Pro-R conformation of NADH are lower and show significantly less variation than the barriers obtained in the case of <i>anti</i> conformation. The effect of basis set and wide range of functionals on the obtained results are also discussed
Phosphorylation as Conformational Switch from the Native to Amyloid State: Trp-Cage as a Protein Aggregation Model
The 20 residue long Trp-cage miniprotein
is an excellent model
for both computational and experimental studies of protein folding
and stability. Recently, great attention emerged to study disease-related
protein misfolding, aggregation, and amyloid formation, with the aim
of revealing their structural and thermodynamic background. Trp-cage
is sensitive to both environmental and structure-modifying effects.
It aggregates with ease upon structure destabilization, and thus it
is suitable for modeling aggregation and amyloid formation. Here,
we characterize the amyloid formation of several sequence modified
and side-chain phosphorylated Trp-cage variants. We applied NMR, circular
dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies,
molecular dynamics (MD) simulations, and transmission electron microscopy
(TEM) in conjunction with thioflavin-T (ThT) fluorescence measurements
to reveal the structural consequences of side-chain phosphorylation.
We demonstrate that the native fold is destabilized upon serine phosphorylation,
and the resultant highly dynamic structures form amyloid-like ordered
aggregates with high intermolecular β-structure content. The
only exception is the D9S(P) variant, which follows an alternative
aggregation process by forming thin fibrils, presenting a CD spectrum
of PPII helix, and showing low ThT binding capability. We propose
a complex aggregation model for these Trp-cage miniproteins. This
model assumes an additional aggregated state, a collagen triple helical
form that can precede amyloid formation. The phosphorylation of a
single serine residue serves as a conformational switch, triggering
aggregation, otherwise mediated by kinases in cell. We show that Trp-cage
miniprotein is indeed a realistic model of larger globular systems
of composite folding and aggregation landscapes and helps us to understand
the fundamentals of deleterious protein aggregation and amyloid formation