Absence of equilibrium cluster phase in concentrated lysozyme solutions

Abstract

In colloidal systems, the interplay between the short range attraction and long-range repulsion can lead to a low density associated state consisting of clusters of individual particles. Recently, such an equilibrium cluster phase was also reported for concentrated solutions of lysozyme at low ionic strength and close to the physiological pH. Stradner et al. [(2004) Equilibrium cluster formation in concentrated protein solutions and colloids. Nature 432:492-495] found that the position of the low-angle interference peak in small-angle x-ray and neutron scattering (SAXS and SANS) patterns from lysozyme solutions was essentially independent of the protein concentration and attributed these unexpected results to the presence of equilibrium clusters. This work prompted a series of experimental and theoretical investigations, but also revealed some inconsistencies. We have repeated these experiments following the protein preparation protocols of Stradner et al. using several batches of lysozyme and exploring a broad range of concentrations, temperature and other conditions. Our measurements were done in multiple experimental sessions at three different high-resolution SAXS and SANS instruments. The lowionic-strength lysozyme solutions displayed a clear shift in peak positions with concentration, incompatible with the presence of the cluster phase but consistent with the system of repulsively interacting individual lysozyme molecules. Within the decoupling approximation, the experimental data can be fitted using an effective interparticle interaction potential involving short-range attraction and long-range repulsion. dynamic arrested state ͉ macromolecular solutions ͉ protein interactions ͉ small-angle scattering ͉ structure factor T he arrested dynamics of colloidal systems and protein solutions interacting via short-range interactions have been actively studied both theoretically and experimentally in recent years (1-4). The mode coupling theory and molecular dynamics (MD) simulations have successfully unified seemingly dissimilar dynamical arrest scenarios in colloidal systems (4, 5). In addition to the conventional glassy state induced by the packing constraints, the presence of short-range attraction leads to a different glassy behavior. The apparently diverse type of dynamical arrest, such as gelation, jamming, glassification or non-ergodicity transition, etc., found in attractive systems can be unified in terms of this attractive glass transition (3). As competing shortrange attraction and long-range repulsion are introduced, additional features are observed (5). In particular, at intermediate volume fractions, the colloidal particles can form an equilibrium cluster phase, which in turn stabilizes a low-density arrested state (6). This type of particle clustering process at low volume fractions has been observed for various colloidal systems (7-9).Although the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory successfully describes the microstructure and equilibrium phase behavior of charged colloidal systems ov...

Do equilibrium clusters exist in concentrated lysozyme solutions?

“…In contrast to our previous findings (1), Shukla et al (2) claim the absence of clusters in concentrated lysozyme solutions. This claim is based on scattering data that show no significant differences from our data, where conditions are similar.…”

“…Furthermore, fits of form and structure factors result in values for the pair potential but cannot provide direct information on the presence of monomers and/or clusters. Nonetheless, for a potential with similar values as determined by Shukla et al (2), MD simulations have confirmed the presence of clusters (3). Therefore, their data (2) actually confirm our earlier data and interpretation (1) that the peak position is concentration-independent and clusters are present in lysozyme solutions, which, as stated in both of our titles, are concentrated.…”

Development of constant-pH simulation methods in implicit solvent and applications in biomolecular systems

“…This can only be done by means of well-converged CpH simulations. Such an observation for LYZ might indicate a promising way to shed light on the understanding of the apparent paradox involved in the LYZ self-association (Shukla et al 2008). …”

Theoretical description of cluster formation in two-Yukawa competing fluids

“…Such peculiar interactions are ubiquitous in soft materials: as few examples, they were used to model protein solutions [1][2][3][4][5], colloids [1,6,7], star polymers [8]. The long-range repulsion is generally attributed to the weakly screened charge carried by the colloidal molecules, or to the presence of cosolutes in the solution [9], whereas the short-range attraction arises from several different mechanisms, including van der Waals interactions, depletion forces, hydrophobic effects [10][11][12].…”