Cosolute modulation of protein oligomerization reactions in the homeostatic timescale.

Protein oligomerization processes are widespread and of crucial importance to understand degenerative diseases and healthy regulatory pathways. One particular case is the homo-oligomerization of folded domains involving domain swapping, often found as a part of the protein homeostasis in the crowded cytosol, composed of a complex mixture of cosolutes. Here, we have investigated the effect of a plethora of cosolutes of very diverse nature on the kinetics of a protein dimerization by domain swapping. In the absence of cosolutes, our system exhibits slow interconversion rates, with the reaction reaching the equilibrium within the average protein homeostasis timescale (24-48 h). In the presence of crowders, though, the oligomerization reaction in the same time frame will, depending on the protein's initial oligomeric state, either reach a pure equilibrium state or get kinetically trapped into an apparent equilibrium. Specifically, when the reaction is initiated from a large excess of dimer, it becomes unsensitive to the effect of cosolutes and reaches the same equilibrium populations as in the absence of cosolute. Conversely, when the reaction starts from a large excess of monomer, the reaction during the homeostatic timescale occurs under kinetic control, and it is exquisitely sensitive to the presence and nature of the cosolute. In this scenario (the most habitual case in intracellular oligomerization processes), the effect of cosolutes on the intermediate conformation and diffusion-mediated encounters will dictate how the cellular milieu affects the domain-swapping reaction.

In silico Design of Phl p 6 Variants With Altered Fold-Stability Significantly Impacts Antigen Processing, Immunogenicity and Immune Polarization.

Introduction: Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. Methods: MAESTRO software was used for in silico prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in E. coli, and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. Results: In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from -5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing in vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. In vivo, stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21. Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

pH-Dependent Protonation of the Phl p 6 Pollen Allergen Studied by NMR and cpH-aMD.

We use state-of-the-art NMR experiments to measure apparent pKa values in the native protein environment and employ a cutting-edge combination of enhanced sampling and constant pH molecular dynamics (MD) simulations to rationalize strong pKa shifts. The major timothy grass pollen allergen Phl p 6 serves as an ideal model system for both methods due to its high number of titratable residues despite its comparably small size. We present a proton transition analysis as intuitive tool to depict the captured protonation state ensemble in atomistic detail. Combining microscopic structural details from MD simulations and macroscopic ensemble averages from NMR shifts leads to a comprehensive view on pH dependencies of protonation states and tautomers. Overall, we find striking agreement between simulation-based pKa predictions and experiment. However, our analyses suggest subtle differences in the underlying molecular origin of the observed pKa shifts. From accelerated constant pH MD simulations, we identify immediate proximity of opposite charges, followed by vicinity of equal charges as major driving forces for pKa shifts. NMR experiments on the other hand, suggest only a weak relation of pKa shifts and close contacts to charged residues, while the strongest influence derives from the dipolar character of α helices. The presented study hence pinpoints opportunities for improvements concerning the theoretical description of protonation state and tautomer probabilities. However, the coherence in the resulting apparent pKa values from simulations and experiment affirms cpH-aMD as a reliable tool to study allergen dynamics at varying pH levels.