The Finding of Nondissolving Lysozyme Crystals and Its Significance for the Study of Hard-to-Crystallize Biological Macromolecules

Abstract

Major discoveries in structural biology depend on obtaining well-diffracting macromolecular crystals. This necessity has motivated many fundamental studies on protein crystallization using lysozyme as a model system. In the present contribution, we report the unprecedented observation of lysozyme crystals that stop dissolving under undersaturated conditions imposed to sub-microliter crystallization drops at mild temperatures. Subsequent growth of the same crystals is apparently undisturbed after the drops are cooled below the saturation temperature. The succession of heating/cooling cycles only partially recovers crystal dissolution while crystal growth becomes gradually slower. Ultimately, increasing and decreasing the temperature between 10 and 37 °C has no visible effect on the size of the crystals. We ascribe this phenomenon to the partial denaturation of the soluble protein in the drop as evidenced by the decreasing glycoside hydrolase activity of lysozyme over the incubation time. The disturbances in the phase transition processes are explained as the result of the changed chemical potential due to different folding states. In a time when the high-hanging fruits in structural biology have to be picked, the present findings call attention to interfacial phenomena as an important, though often imperceptible, aspect that affects protein stability and justifies further optimization of current crystallization methods.

The nucleation of protein crystals as a race against time with on- and off-pathways

“…Only conditions of uncertain crystallization outcome (for which the success rates were different from 0 or 100%) were chosen. As in a previously reported control analysis, 0.7 ml crystallization drops under comparable experimental conditions showed no major variation of protein concentration and enzymatic activity during the first 24 h of incubation (Ferreira et al, 2016).…”

“…has not been considered in the schematic mechanism in Fig. 7, concentration gradients created close to the boundary regions can promote heterogeneous nucleation (Martins et al, 2008), alter the local effective concentrations of protein and precipitant (García-Ruiz et al, 2016), and/or accelerate protein loss by denaturation (Ferreira et al, 2016). Liquid-liquid separation occurring at higher supersaturation levels (Muschol & Rosenberger, 1997;Vekilov & Vorontsova, 2014;Pan et al, 2010) is also absent from the simplified crystallization model represented in Fig.…”

MIRRAGGE – Minimum Information Required for Reproducible AGGregation Experiments

“…This is acchieved by including a flexible field of "additional key information" at the end of the tabular descriptions of the purification protocol and of the aggregation assay, wherein relevant remarks concerning the aggregation state of the protein, sample collection procedures in gel filtration chromatography, the occurrence of contaminants or co-solvents, the procedure adopted for removal of air bubbles, a critical sequence of reagent addition, etc., can be emphasized. A comprehensive characterization of the aggregation assay in terms of the total volume of reaction, plate/cuvette/vial geometry and material, method of evaporation control, size and material of beads (if present), and type of agitation is required on account of the effects of interfaces and shear flow on protein aggregation (Giehm and Otzen, 2010;Bekard et al, 2011;Yoshimura et al, 2012;Ferreira et al, 2016;Koepf et al, 2018). As a major determinant of phase separation, protein concentration is discriminated (i) before storage of the purified protein, (ii) immediately before aggregation, e.g., after the final filtration step, and (iii) during aggregation.…”

Chemical Kinetic Strategies for High‐Throughput Screening of Protein Aggregation Modulators

“…[42] Changing the ionic strength of amyloidogenic solutionsb y the addition (or removal)ofs alts can have adramatic influence on amyloid aggregation in vitro, [39] which is understood in terms of the role of solubility in determining the rate and extent of protein self-assembly. [7,8,12,40] These modulation effects are considered "apparent" (Figure 3f)s incet hey are unspecific and tend to be buffered in cells and tissues media. Apparenti nhibitors that solely alter the activity coefficient of the protein will originate superimposed progress curvesi fr epresented in normalizedT hT fluorescenceu nits (Figure 3f, bottom); this has been previously illustrated using published data of Pronchik et al [41] on the influence of hydrophobic interfaces on a-synuclein aggregation.…”