Patterns of protein–protein interactions in salt solutions and implications for protein crystallization

Abstract

The second osmotic virial coefficients of seven proteins-ovalbumin, ribonuclease A, bovine serum albumin, a-lactalbumin, myoglobin, cytochrome c, and catalase-were measured in salt solutions.Comparison of the interaction trends in terms of the dimensionless second virial coefficient b 2 shows that, at low salt concentrations, protein-protein interactions can be either attractive or repulsive, possibly due to the anisotropy of the protein charge distribution. At high salt concentrations, the behavior depends on the salt: In sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations, whereas in ammonium sulfate, proteins show a sharp drop in b 2 with increasing salt concentration beyond a particular threshold. The experimental phase behavior of the proteins corroborates these observations in that precipitation always follows the drop in b 2 . When the proteins crystallize, they do so at slightly lower salt concentrations than seen for precipitation. The b 2 measurements were extended to other salts for ovalbumin and catalase. The trends follow the Hofmeister series, and the effect of the salt can be interpreted as a water-mediated effect between the protein and salt molecules. The b 2 trends quantify protein-protein interactions and provide some understanding of the corresponding phase behavior. The results explain both why ammonium sulfate is among the best crystallization agents, as well as some of the difficulties that can be encountered in protein crystallization. Keywords: protein interactions; protein crystallization; osmotic second virial coefficient; self-interaction chromatographyThe relation between the molecular structure of a protein and the interaction potential between two such molecules is the foundation of a thermodynamic approach to understanding and predicting the behavior of protein solutions.Ultimately, such a relation enables calculation of the phase behavior and thus understanding of the formation of different phases, such as crystals, dense liquid phases, gels, and aggregates. In practice, the interactions between proteins in solutions are modulated by additives such as salts, hydrophilic polymers, or small organic molecules that may be present in formulations or added to induce crystallization. The focus of the present work is limited to exploring the role of different salts in tuning protein interactions.The interactions between proteins include contributions from at least electrostatic and van der Waals forces, hydration effects, hydrogen bonding, salt bridging, and Reprint requests to: Abraham M.

Electrochemical behaviour of myoglobin at an array of microscopic liquid–liquid interfaces

“…Salt ions are categorised according to whether they denature or stabilise a protein [47]. At high salt concentrations, protein-protein interactions become favourable over electrostatic repulsion and this is the driving force for precipitation [48,49] and aggregation [50]. It has also been reported that at low pH, protein-protein interactions become more favourable [51].…”

Self-association of a highly charged arginine-rich cell-penetrating peptide

“…Both electrostatic adhesive and double-layer forces are weakened by the presence of salt in the solution. As a nontrivial consequence, the propensity of the proteins to aggregate is heightened at low-to-intermediate ionic strengths (1)(2)(3)6). This is because of lowering of Coulombic repulsion due to salt screening of the net charge of the protein, in conjunction with the presence of the adhesive force, which operates at shorter distances and is therefore less efficiently screened.…”

Halorhodopsin pumps Cl–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

“…These studies shed light on how ion transfer mechanisms are controlled through the interplay of protein and ion electrostatics. I ons bind to proteins to carry out many essential biological reactions, including muscle function (1), signal transduction (2), protein-protein interactions (3), and oxygen transport (4). The transmembrane electrochemical gradient generated by ion and proton pumps drives key processes such as ATP synthesis and nerve transmission (5).…”