Protein condensates play essential roles in physiological processes and pathological conditions. Recently discovered mesoscopic protein-rich clusters may act as crucial precursors for the nucleation of ordered protein solids, such as crystals, sickle hemoglobin polymers, and amyloid fibrils. These clusters challenge settled paradigms of protein condensation as the constituent protein molecules present features characteristic of both partially misfolded and native proteins. Here we employ the antimicrobial enzyme lysozyme and examine the similarities between mesoscopic clusters, amyloid structures, and disordered aggregates consisting of chemically modified protein. We show that the mesoscopic clusters are distinct from the other two classes of aggregates. Whereas cluster formation and amyloid oligomerization are both reversible, aggregation triggered by reduction of the intramolecular S-S bonds is permanent. In contrast to the amyloid structures, protein molecules in the clusters retain their enzymatic activity. Furthermore, an essential feature of the mesoscopic clusters is their constant radius of less than 50 nm. The amyloid and disordered aggregates are significantly larger and rapidly grow. These findings demonstrate that the clusters are a product of limited protein structural flexibility. In view of the role of the clusters in the nucleation of ordered protein solids, our results suggest that fine-tuning the degree of protein conformational stability is a powerful tool to control and direct the pathways of protein condensation.

According to recently proposed two-step nucleation mechanisms, crystal nuclei form within preexisting dense liquid clusters. Clusters with radii about 100 nm, which capture from 10(-7) to 10(-3) of the total protein, have been observed with numerous proteins and shown to host crystal nucleation. Theories aiming to understand the mesoscopic size and small protein fraction held in the clusters have proposed that in solutions of single-chain proteins, the clusters consist of partially misfolded protein molecules. To test this conjecture, we perturb the protein conformation by shearing solutions of the protein lysozyme. We demonstrate that shear rates greater than a threshold applied for longer than 1 h reduce the volume of the cluster population. The likely mechanism of the observed response involves enhanced partial unfolding of lysozyme molecules, which exposes hydrophobic surfaces between the constituent domains to the aqueous solution.

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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.

Adherens junctions (AJs) are a defining feature of all epithelial cells. They regulate epithelial tissue architecture and integrity, and their dysregulation is a key step in tumor metastasis. AJ remodeling is crucial for cancer progression, and it plays a key role in tumor cell survival, growth, and dissemination. Few studies have examined AJ remodeling in cancer cells consequently, it remains poorly understood and unleveraged in the treatment of metastatic carcinomas. Fascin1 is an actin‐bundling protein that is absent from the normal epithelium but its expression in colon cancer is linked to metastasis and increased mortality. Here, we provide the molecular mechanism of AJ remodeling in colon cancer cells and identify for the first time, fascin1's function in AJ remodeling. We show that in colon cancer cells fascin1 remodels junctional actin and actomyosin contractility which makes AJs less stable but more dynamic. By remodeling AJs fascin1 drives mechanoactivation of WNT/β‐catenin signaling and generates “collective plasticity” which influences the behavior of cells during cell migration. The impact of mechanical inputs on WNT/β‐catenin activation in cancer cells remains poorly understood. Our findings highlight the role of AJ remodeling and mechanosensitive WNT/β‐catenin signaling in the growth and dissemination of colorectal carcinomas.