Vincent Vagenende

Mechanisms of Protein Stabilization and Prevention of Protein Aggregation by Glycerol

The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Glycerol is known to shift the native protein ensemble to more compact states. Glycerol also inhibits protein aggregation during the refolding of many proteins. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. In this study, we derive mechanisms of glycerol-induced protein stabilization by combining the thermodynamic framework of preferential interactions with molecular-level insight into solvent-protein interactions gained from molecular simulations. Contrary to the common conception that preferential hydration of proteins in polyol/water mixtures is determined by the molecular size of the polyol and the surface area of the protein, we present evidence that preferential hydration of proteins in glycerol/water mixtures mainly originates from electrostatic interactions that induce orientations of glycerol molecules at the protein surface such that glycerol is further excluded. These interactions shift the native protein toward more compact conformations. Moreover, glycerol preferentially interacts with large patches of contiguous hydrophobicity where glycerol acts as an amphiphilic interface between the hydrophobic surface and the polar solvent. Accordingly, we propose that glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol. These mechanisms agree well with experimental data available in the literature, and we discuss the extent to which these mechanisms apply to other cosolvents, including polyols, arginine, and urea.

Protein-associated cation clusters in aqueous arginine solutions and their effects on protein stability and size.

Arginine is one of the most prominent residues in protein interactions, and arginine hydrochloride is widely used as an additive in protein solutions because of its exceptional effects on protein association and folding. The molecular origins of arginine effects on protein processes remain, however, controversial, and little is known about the molecular interactions between arginine cations and protein surfaces in aqueous arginine solutions. In this study, we report a unique biochemical phenomenon whereby clusters of arginine cations (Arg(+)) are associated with a protein surface. The formation of protein-associated Arg(+) clusters is initiated by Arg(+) ions that associate with specific protein surface loci through cooperative interactions with protein guanidinium and carboxyl groups. Molecular dynamics simulations indicate that protein-associated Arg(+) ions subsequently attract other Arg(+) ions and form dynamic cation clusters that extend further than 10 Å from the protein surface. The effects of arginine on the thermal stability and size of lysozyme and ovalbumin are measured over a wide concentration range (0 to 2 M), and we find that the formation of protein-associated Arg(+) clusters consistently explains the complex effects of arginine on protein stability and size. This study elucidates the molecular mechanisms and implications of cluster formation of Arg(+) ions at a protein surface, and the findings of this study may be used to manipulate synthetic and biological systems through arginine-derived groups.

Molecular anatomy of preferential interaction coefficients by elucidating protein solvation in mixed solvents: methodology and application for lysozyme in aqueous glycerol.

Preferential interaction coefficients of proteins in mixed solvents are bulk thermodynamic parameters that relate molecular characteristics of protein solvation with solvent effects on protein thermodynamics. Because of their bulk nature, they give no insight in the molecular level nature of protein solvation. In this study, we develop a methodology which provides insight into the molecular anatomy of preferential interaction coefficients by elucidating protein solvation in mixed solvents. Our methodology makes use of molecular simulations and reveals intricacies of solvent-protein interactions which are not accounted for by less detailed models for solvent effects on protein thermodynamics. This is demonstrated for lysozyme in 30 vol % aqueous glycerol. We find that solvent regions near protein O- and N-atoms that favor the formation of multiple hydrogen-bonds with glycerol positively contribute to the preferential interaction coefficient (15+/-4) due to the preferential solvation by glycerol molecules with long residence times (>2 ns). Yet, the overall value of the preferential interaction coefficient is negative as solvent regions near protein surface loci with similar affinities for glycerol and water have a stronger negative contribution (-22+/-4). On the basis of these results, we discuss the current scope and future prospects of our methodology to understand solvent effects on protein thermodynamics.

Integrative analysis workflow for the structural and functional classification of C-type lectins

BackgroundIt is important to understand the roles of C-type lectins in the immune system due to their ubiquity and diverse range of functions in animal cells. It has been observed that currently confirmed C-type lectins share a highly conserved domain known as the C-type carbohydrate recognition domain (CRD). Using the sequence profile of the CRD, an increasing number of putative C-type lectins have been identified. Hence, it is highly needed to develop a systematic framework that enables us to elucidate their carbohydrate (glycan) recognition function, and discover their physiological and pathological roles.ResultsPresented herein is an integrated workflow for characterizing the sequence and structural features of novel C-type lectins. Our workflow utilizes web-based queries and available software suites to annotate features that can be found on the C-type lectin, given its amino acid sequence. At the same time, it incorporates modeling and analysis of glycans - a major class of ligands that interact with C-type lectins. Thereafter, the results are analyzed together with context-specific knowledge to filter off unlikely predictions. This allows researchers to design their subsequent experiments to confirm the functions of the C-type lectins in a systematic manner.ConclusionsThe efficacy and usefulness of our proposed immunoinformatics workflow was demonstrated by applying our integrated workflow to a novel C-type lectin -CLEC17A - and we report some of its possible functions that warrants further validation through wet-lab experiments.

Allantoin as a solid phase adsorbent for removing endotoxins.

In this study we present a simple and robust method for removing endotoxins from protein solutions by using crystals of the small-molecule compound 2,5-dioxo-4-imidazolidinyl urea (allantoin) as a solid phase adsorbent. Allantoin crystalline powder is added to a protein solution at supersaturated concentrations, endotoxins bind and undissolved allantoin crystals with bound endotoxins are removed by filtration or centrifugation. This method removes an average of 99.98% endotoxin for 20 test proteins. The average protein recovery is ∼80%. Endotoxin binding is largely independent of pH, conductivity, reducing agent and various organic solvents. This is consistent with a hydrogen-bond based binding mechanism. Allantoin does not affect protein activity and stability, and the use of allantoin as a solid phase adsorbent provides better endotoxin removal than anion exchange, polymixin affinity and biological affinity methods for endotoxin clearance.

Amide-mediated hydrogen bonding at organic crystal/water interfaces enables selective endotoxin binding with picomolar affinity.

Since the discovery of endotoxins as the primary toxic component of Gram-negative bacteria, researchers have pursued the quest for molecules that detect, neutralize, and remove endotoxins. Selective removal of endotoxins is particularly challenging for protein solutions and, to this day, no general method is available. Here, we report that crystals of the purine-derived compound allantoin selectively adsorb endotoxins with picomolar affinity through amide-mediated hydrogen bonding in aqueous solutions. Atom force microscopy and chemical inhibition experiments indicate that endotoxin adsorption is largely independent from hydrophobic and ionic interactions with allantoin crystals and is mediated by hydrogen bonding with amide groups at flat crystal surfaces. The small size (500 nm) and large specific surface area of allantoin crystals results in a very high endotoxin-binding capacity (3 × 10(7) EU/g) which compares favorably with known endotoxin-binding materials. These results provide a proof-of-concept for hydrogen bond-based molecular recognition processes in aqueous solutions and establish a practical method for removing endotoxins from protein solutions.

Self-assembly of lipopolysaccharide layers on allantoin crystals

Self-assembly of lipopolysaccharides (LPS) on solid surfaces is important for the study of bacterial membranes, but has not been possible due to technical difficulties and the lack of suitable solid supports. Recently we found that crystals of the natural compound allantoin selectively bind pure LPS with sub-nanomolar affinity. The physicochemical origins of this selectivity and the adsorption mode of LPS on allantoin crystals remain, however, unknown. In this study we present evidence that LPS adsorption on allantoin crystals is initiated through hydrogen-bond attachment of hydrophilic LPS regions. Hydrophobic interactions between alkyl chains of adjacently adsorbed LPS molecules subsequently promote self-assembly of LPS layers. The essential role of hydrogen-bond interactions is corroborated by our finding that allantoin crystals bind to practically any hydrophilic surface chemistry. Binding contributions of hydrophobic interactions between LPS alkyl chains are evidenced by the endothermic nature of the adsorption process and explain why the binding affinity for LPS is several orders of magnitude higher than for proteins (lysozyme, BSA and IgG) and polysaccharides. Self-assembly of LPS layers via hydrogen-bond attachment on allantoin crystals emerges as a novel binding mechanism and could be considered as a practical method for preparing biomimetic membranes on a solid support.

Quantifying the Molecular Origins of Opposite Solvent Effects on Protein-Protein Interactions

Although the nature of solvent-protein interactions is generally weak and non-specific, addition of cosolvents such as denaturants and osmolytes strengthens protein-protein interactions for some proteins, whereas it weakens protein-protein interactions for others. This is exemplified by the puzzling observation that addition of glycerol oppositely affects the association constants of two antibodies, D1.3 and D44.1, with lysozyme. To resolve this conundrum, we develop a methodology based on the thermodynamic principles of preferential interaction theory and the quantitative characterization of local protein solvation from molecular dynamics simulations. We find that changes of preferential solvent interactions at the protein-protein interface quantitatively account for the opposite effects of glycerol on the antibody-antigen association constants. Detailed characterization of local protein solvation in the free and associated protein states reveals how opposite solvent effects on protein-protein interactions depend on the extent of dewetting of the protein-protein contact region and on structural changes that alter cooperative solvent-protein interactions at the periphery of the protein-protein interface. These results demonstrate the direct relationship between macroscopic solvent effects on protein-protein interactions and atom-scale solvent-protein interactions, and establish a general methodology for predicting and understanding solvent effects on protein-protein interactions in diverse biological environments.