Weakly-bound Dimers that Underlie the Crystal Nucleation Precursors in Lysozyme Solutions

Abstract

Protein crystallization is central to understanding of molecular structure in biology, a vital part of processes in the pharmaceutical industry, and a crucial component of numerous disease pathologies. Crystallization starts with nucleation and how nucleation proceeds determines the crystallization rate and essential properties of the resulting crystal population. Recent results with several proteins indicate that crystals nucleate within preformed mesoscopic protein-rich clusters. The origin of the mesoscopic clusters is poorly understood. In the case of lysozyme, a common model of protein biophysics, earlier findings suggest that clusters exist owing to the dynamics of formation and decay of weakly-bound transient dimers. Here we present evidence of a weakly bound lysozyme dimer in solutions of this protein. We employ two electrospray mass spectrometry techniques, a combined ion mobility separation mass spectrometry and a high-resolution implementation. To enhance the weak but statistically-significant dimer signal we develop a method based on the residuals between the maxima of the isotope peaks in Fourier space and their Gaussian envelope. We demonstrate that these procedures sensitively detect the presence of a noncovalently bound dimer and distinguish its signal from other polypeptides, noise, and sampling artefacts. These findings contribute essential elements of the crystal nucleation mechanism of lysozyme and other proteins and suggest pathways to control nucleation and crystallization by enhancing or suppressing weak oligomerization.

Effects of Protein Unfolding on Aggregation and Gelation in Lysozyme Solutions

“…Our results resonate with the observations of previous investigators on aggregation phenomena in lysozyme solutions. The importance of driving forces for aggregation at physiological temperatures was emphasized in the work of Vekilov et al [23], where the presence of transient dimers of lysozyme was found. Also, according to Strander et al [10], at the same ionic strength and pH, the presence of short-range attractions and long-range repulsions leads to the formation of dense liquid phases in lysozyme solutions.…”

“…It was found that these aggregates typically had a diameter between 60-200 nm and a fraction of~10 −4 of total soluble protein [21]. The aggregates were commonly thought to be reversible [22], undergoing dynamic molecular exchange with protein in solution [23].…”