Probing pattern and dynamics of disulfide bridges using synthesis and NMR of an ion channel blocker peptide toxin with multiple diselenide bonds

Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin containing four disulfide bridges, is a high affinity blocker of the voltage-gated potassium channel Kv1.3, but also blocks Kv1.2. To improve potential therapeutic use of the toxin, we have designed a double substituted analog, [N17A/F32T]-AnTx, which showed comparable Kv1.3 affinity to the wild-type peptide, but also a 2500-fold increase in the selectivity for Kv1.3 over Kv1.2. In the present study we have achieved the chemical synthesis of a Sec-analog in which all cysteine (Cys) residues have been replaced by selenocysteine (Sec) forming four diselenide bonds. To the best of our knowledge this is the first time to replace, by chemical synthesis, all disulfide bonds with isosteric diselenides in a peptide/protein. Gratifyingly, the key pharmacological properties of the Sec-[N17A/F32T]-AnTx are retained since the peptide is functionally active. We also propose here a combined experimental and theoretical approach including NOE- and Se-77-based NMR supplemented by MD simulations for conformational and dynamic characterization of the Sec-[N17A/F32T]-AnTx. Using this combined approach allowed us to attain unequivocal assignment of all four diselenide bonds and supplemental MD simulations allowed characterization of the conformational dynamics around each disulfide/diselenide bridge

Solvating atomic level fine-grained proteins in supra-molecular level coarse-grained water for molecular dynamics simulations

Simulation of the dynamics of a protein in aqueous solution using an atomic model for both the protein and the many water molecules is still computationally extremely demanding considering the time scale of protein motions. The use of supra-atomic or supra-molecular coarse-grained (CG) models may enhance the computational efficiency, but inevitably at the cost of reduced accuracy. Coarse-graining solvent degrees of freedom is likely to yield a favourable balance between reduced accuracy and enhanced computational speed. Here, the use of a supra-molecular coarse-grained water model that largely preserves the thermodynamic and dielectric properties of atomic level fine-grained (FG) water in molecular dynamics simulations of an atomic model for four proteins is investigated. The results of using an FG, a CG, an implicit, or a vacuum solvent environment of the four proteins are compared, and for hen egg-white lysozyme a comparison to NMR data is made. The mixed-grained simulations do not show large differences compared to the FG atomic level simulations, apart from an increased tendency to form hydrogen bonds between long side chains, which is due to the reduced ability of the supra-molecular CG beads that represent five FG water molecules to make solvent-protein hydrogen bonds. But, the mixed-grained simulations are at least an order of magnitude faster than the atomic level one

Molecular determinants of nonaqueous biocatalysis

Dissertation presented to obtain the Ph.D degree in BiochemistryOver the last thirty years, the tremendous biotechnological potential of nonaqueous biocatalysis has boosted research efforts in this area. Numerous studies have tried to elucidate how enzymes work in these nonconventional media and many properties are now well understood. However, when this thesis was initiated, some aspects of this field were poorly characterized at the molecular level. In particular, the molecular determinants of protein-ion interactions, enzyme stability, and molecular memory, are important issues which were lacking a thorough molecular analysis. These three subjects are herein investigated using molecular simulation methodologies.(...

From Solution Into the Gas Phase: Studying Protein Hydrogen Exchange and Electrospray Ionization Using Molecular Dynamics Simulation

Here, we apply Molecular Dynamics (MD) simulations to investigate fundamental aspects of structural mass spectrometry (MS). We first examine microscopic phenomena underlying Hydrogen/Deuterium exchange (HDX). HDX interrogates structural dynamics of proteins by measuring the rate of Deuterium uptake into backbone amides. We perform microsecond MD simulations on ubiquitin to investigate this process. We find that HDX protection often cannot be explained by H‑bonding or solvent accessibility considerations. These findings caution against non-critical use of HDX data in structural contexts. We next use MD to examine the Electrospray ionization (ESI) mechanism of proteins. ESI is a soft ionization technique resulting in the production of gaseous protein ions. The mechanism of ion formation from nanometer sized droplets is unclear. We apply a trajectory stitching MD approach to simulate protein-containing nanodroplets, finding that natively‑folded proteins remain solvated as droplets shrink. Residual charge carriers remain following desolvation, consistent with Dole’s charged residue model

Molecular modeling study of β-lactoglobulin dimerization: a first step to hypoallergen design for immunotherapy

"Milk and its derivatives are important worldwide food sources, particularly for infant nutrition, but face a major health complication: some of their proteins are allergens, especially β-lactoglobulin (BLG), a major component of bovine milk. The fate of BLG upon ingestion remains unsettled, being unclear how extensive BLG proteolysis is and how it affects allergenicity. The fact that proteolytic resistance and antigenic response remain related even for non-oral administration suggests that they are not causally related but rather reflect an underlying common feature.(...)

Electrostatic screening in molecular dynamics simulations

The screened Coulombic potential has been shown to describe satisfactorily equilibrium properties like pK shifts, the effects of charged groups on redox potentials and binding constants of metal ions. To test how well the screening of the electrostatic potential describes the dynamical trajectory of a macromolecular system, a series of comparative simulations have been carried out on a protein system which explicitly included water molecules and a system in vacuo. For the system without solvent the results of using (i) the standard potential form were compared with results of (ii) the potential where the Coulomb term was modified by the inclusion of a distance dependent dielectric, ε(r), to model the screening effect of bulk water, and (iii) standard potential modified by reducing the charge on ionized residue side chains. All molecular dynamics simulations have been carried out on bovine pancreatic trypsin inhibitor. Comparisons between the resulting trajectories, averaged structures, hydrogen bonding patterns and properties such as solvent accessible surface area and radius of gyration are described. The results show that the dynamical behaviour of the protein calculated with a screened electrostatic term compares more favourably with the time-dependent structural changes of the full system with explicitly included water than the standard vacuum simulatio