Direct Hydride Shift Mechanism and Stereoselectivity of P450_nor Confirmed by QM/MM Calculations

Nitric oxide reductase (P450_nor) found in Fusarium oxysporum catalyzes the reduction of nitric oxide to N₂O 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