Effect of diffusion kinetics on the ice nucleation temperature distribution

The nucleation behavior of water is crucial in many fields, spanning meteorology, glaciology, biology, and astrophysics. We report observations suggesting an effect of diffusion kinetics in water on the heterogeneous immersion/contact mode nucleation temperature distribution of ice. We performed differential scanning calorimetry analyses of repeated freeze/thaw cycles and investigated the effect of several variables on the regularity of the nucleation temperature distributions obtained. We observed that the thawing temperature and residence time above 0 degrees C affect the width of the measured distributions. We explain the observed phenomena according to the diffusion behavior of an external nucleator. Specifically, conditions of enhanced diffusion of the nucleator translated into broader, more scattered distributions, while conditions of limited diffusion translated into narrower, more regular distributions. Lastly, based on our experimental findings, we propose a theoretical explanation centered on the temperature dependence of diffusion kinetics in water

Rational design of freeze-drying formulations: a molecular dynamics approach

The freezing step plays a fundamental role in the freeze drying process, as it determines product morphology and overall efficiency. The current approach to the selection of freezing conditions is however non-systematic, resulting in poor process control. Here we show how mathematical models, and a design space approach, can guide the selection of the optimal freezing protocol, focusing on both process performance and protein stability

Rational design of freeze-drying formulations: a Molecular Dynamics approach

Even though freeze-drying is widely used for the preparation of biopharmaceuticals, it causes stresses which can result in unfolding or aggregation. Suitable excipients must therefore be added to avoid loss of activity. However, at present, the choice of a suitable formulation is mainly empirical, due to a lack of knowledge about the phenomena involved. In this context, the objective of the present work was to understand the molecular mechanisms at the basis of protein stabilization, and to guide the choice of suitable excipients

A new transfer free energy based implicit solvation model for the description of disordered and folded proteins

Most biological events occur on time scales that are difficult to access using conventional all-atom molecular dynamics simulations in explicit solvent. Implicit solvent techniques offer a promising solution to this problem, alleviating the computational cost associated with the simulation of large systems and accelerating the sampling compared to explicit solvent models. The substitution of water molecules by a mean field, however, introduces simplifications that may penalize accuracy and impede the prediction of certain physical properties. We demonstrate that existing implicit solvent models developed using a transfer free energy approach, while satisfactory at reproducing the folding behavior of globular proteins, fare less well in characterizing the conformational properties of intrinsically disordered proteins. We develop a new implicit solvent model that maximizes the degree of accuracy for both disordered and folded proteins. We show, by comparing the simulation outputs to experimental data, that in combination with the a99SB-disp force field, the implicit solvent model can describe both disordered (a beta 40, PaaA2, and drkN SH3) and folded ((AAQAA)(3), CLN025, Trp-cage, and GTT) peptides. Our implicit solvent model permits a computationally efficient investigation of proteins containing both ordered and disordered regions, as well as the study of the transition between ordered and disordered protein states. implicit solvent + a99SB-disp force field disordered proteins: 440 PaaA2 drkN SH3 20 a) `p 15 U 10 5 0 optimized approach appl app2 app3 app4 disordere folded & BULL; total fast-folding peptides: (AAQAA)3 CLN025 Trp-cage GT

Effect of Surfactants on Surface-Induced Denaturation of Proteins: An Insight from Molecular Dynamics

Proteins are marginally stable, and when they bind to interfaces the resulting conformational changes can lead to loss of biological activity. In order to stabilize proteins in experiments where surface-induced denaturation is an issue, surfactants are commonly used. [1] However, the mechanism by which they prevent surface-induced denaturation of proteins is not completely understood. In the present work, the folding of the GB1 hairpin (shown in Fig. 1a) at the air-water, silica-water and ice-water surfaces is investigated, in the presence and absence of the surfactant Tween 80. Atomistic molecular dynamics (MD) simulations, coupled with the enhanced sampling method known as parallel bias metadynamics (PBMetaD) [2], are used for this investigation. Our simulations reveal that GB1 is destabilized at the air-water and ice-water interfaces, but stabilized at the silica surface. Tween 80 stabilizes the protein at the air-water and ice-water surfaces (Fig. 1b), but slightly destabilizes the protein at the silica interface. The surfactant molecules bind to the air and silica surface, while they cluster around the protein in the case of ice. An orientation-dependent mechanism of the surfactants is also active, in which the protein is stabilized when the hydrophilic heads of the surfactant are oriented towards the protein, and destabilized when the hydrophobic tails point towards the peptide. The latter orientation stabilizes partially unfolded states of the protein, characterized by a larger non-polar surface area. The tails-toward-the-protein configuration is favored in a hydrophilic environment, explaining the mild destabilization observed at the silica-water interface. By contrast, the ice-water surface promotes the heads-toward-the-protein arrangement, that stabilizes the protein native structure. Finally, in the case of the air-water interface, the coating of the interface by the surfactant molecules, and the resulting inhibition of protein adsorption, accounts for the observed stabilization of the protein native structure. [3

Force Field Parameterization for the Description of the Interactions between Hydroxypropyl-β-Cyclodextrin and Proteins

Cyclodextrins are cyclic oligosaccharides, widely used as drug carriers, solubilizers, and excipients. Among cyclodextrins, the functionalized derivative known as hydroxypropyl-β-cyclodextrin (HPβCD) offers several advantages due to its unique structural features. Its optimal use in pharmaceutical and medical applications would benefit from a molecular-level understanding of its behavior, as can be offered by molecular dynamics simulations. Here, we propose a set of parameters for all-atom simulations of HPβCD, based on the ADD force field for sugars developed in our group, and compare it to the original CHARMM36 description. Using Kirkwood-Buff integrals of binary HPβCD-water mixtures as target experimental data, we show that the ADD-based description results in a considerably improved prediction of HPβCD self-association and interaction with water. We then use the new set of parameters to characterize the behavior of HPβCD toward the different amino acids. We observe pronounced interactions of HPβCD with both polar and nonpolar moieties, with a special preference for the aromatic rings of tyrosine, phenylalanine, and tryptophan. Interestingly, our simulations further highlight a preferential orientation of HPβCD's hydrophobic cavity toward the backbone atoms of amino acids, which, coupled with a favorable interaction of HPβCD with the peptide backbone, suggest a propensity for HPβCD to denature proteins

Surface-driven denaturation of proteins during freeze-drying: An insight into the role of surfactants

Protein-based therapeutics may bind to interfaces during the freeze-drying process, possibly resulting in loss of activity. Here we investigate the mechanism by which surfactant molecules can counteract surface-induced denaturation through a detailed study of the stability of the GB1 peptide at the air-water, ice-water and silica-water interfaces. Using molecular dynamics simulations coupled with metadynamics we show that the amphiphilic nature of surfactants is key to their stabilizing/destabilizing effect, with an orientation-dependent mechanism in which the protein is stabilized when the hydrophilic heads of the surfactant point toward the protein

A proximity-based in silico approach to identify redox-labile disulfide bonds: The example of FVIII

Allosteric disulfide bonds permit highly responsive, transient 'switch-like' properties that are ideal for processes like coagulation and inflammation that require rapid and localised responses to damage or injury. Haemophilia A (HA) is a rare bleeding disorder managed with exogenous coagulation factor(F) VIII products. FVIII has eight disulfide bonds and is known to be redox labile, but it is not known how reduction/oxidation affects the structure-function relationship, or its immunogenicity-a serious complication for 30% severe HA patients. Understanding how redox-mediated changes influence FVIII can inform molecular engineering strategies aimed at improving activity and stability, and reducing immunogenicity. FVIII is a challenging molecule to work with owing to its poor expression and instability so, in a proof-of-concept study, we used molecular dynamics (MD) to identify which disulfide bonds were most likely to be reduced and how this would affect structure/function; results were then experimentally verified. MD identified Cys1899-Cys1903 disulfide as the most likely to undergo reduction based on energy and proximity criteria. Further MD suggested this reduction led to a more open conformation. Here we present our findings and highlight the value of MD approaches