Do protein crystals nucleate within dense liquid clusters?

Abstract

Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10 À3 of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein-rich clusters in lysozyme and glucose isomerase solutions are locations for crystal nucleation.

Two types of amorphous protein particles facilitate crystal nucleation

“…From these observations, we conclude that the particles are amorphous solid particles (ASPs) of lysozyme. The ASPs are similar in size to the mesoscopic clusters, which have been suggested to be precursors for crystal nucleation, as reported in previous studies (9,10). However, crystals never formed within the ASPs during our observations.…”

“…The latter nature of the precursor differs from the stable macroscopically dense liquid formed as a result of the liquid-liquid phase separation (8). Such protein-rich mesoscopic clusters have been observed for many proteins, primarily using optical techniques, and have been tentatively identified as precursors for crystal nucleation (9)(10)(11)(12). Several important questions concerning this mechanism remain unanswered.…”

A Single Spherical Assembly of Protein Amyloid Fibrils Formed by Laser Trapping

“…Dynamics and mechanism of amyloid formation and crystallization of proteins have been studied comparatively, since both consist of nucleation and the following growth process where some time lag is usually observed. [1] Amyloid nucleation is started from misfolded and unfolded conformations of proteins and leads to fibrillation through mutual interactions of prefibrillar oligomer intermediates. [2] Protein crystallization is coupled with the formation of protein clusters containing solvent waters and the subsequent generation of ah ighly concentrated area of such clusters.…”

“…[2] Protein crystallization is coupled with the formation of protein clusters containing solvent waters and the subsequent generation of ah ighly concentrated area of such clusters. [3] Nucleation and the crystal growth proceed in such an area of af ew tens to af ew hundreds of nanometers.M ost of the experiments for amyloid formation and crystallization are carried out in solution by tuning pH, [1] salt concentration, [1] and temperature [2] and by applying ultrasonication, [3] electro-magnetic field, [4] and pulsed laser irradiation. [5,6] Therefore, all the processes of nucleation and growth of amyloid and crystal in solution proceed randomly in parallel, whose dynamic evolutions are monitored and analyzed as an ensemble of amyloid fibrils or crystals.I ti sc onsidered very promising to propose an ew experimental approach for preparing as ingle spherical assembly of protein amyloid, analyzing its dynamics,and fabricating micro-structures from single assemblies.Itwill enable us to perform amyloid studies by watching always when and where individual assemblies of amyloid fibrils are prepared, monitored, and utilized.…”

Phase separation: Bridging polymer physics and biology

“…For instance, one current hypothesis is that liquid-like condensates serve to facilitate reactions by concentrating enzymes and substrates, while solid-like granules can more significantly sequester enzymes away from their targets [6,29] and/or potentially serve as a filter to prevent the passage of certain molecules, as in the case of the nuclear pore complex [92,93]. However, some condensed liquid states have shown metastability, and will age over time to form an aggregated, amyloid-like, or even crystalline state [90,[94][95][96][97]. Thus, while the formation of more solid-like structures can drive cell function, it has been proposed that misregulation of the pathways associated with these materials may be relevant to neurodegenerative and other protein aggregation-related diseases [4,29,35,89,90,[97][98][99].…”