Opportunistic complexation and mesoscopic aggregates in protein solutions

University Of Houston, Houston TX

Investigators

Abstract

Title: Opportunistic complexation and mesoscopic aggregates in protein solution Protein aggregation is a central problem of biophysics and other life sciences. This investigation focuses on a particularly puzzling type of protein aggregation during which protein-rich inclusions form in protein solutions. These inclusions are a micron or less in size and have been called the "mesoscopic clusters". Despite their small volume, the clusters are essential nucleation sites for ordered protein solids such as crystals and sickle cell anemia fibers. The mesoscopic clusters are also at odds with standard notions of thermodynamics, which dictates that such clusters should be either much larger or should not exist at all. Understanding the molecular origin of the mesoscopic clusters will resolve a major fundamental question of thermodynamics. This research will test the hypothesis that the mesoscopic clusters are caused by formation of long-lived complexes made up of individual protein molecules. A combination of advanced experimental techniques, theoretical modeling, and computer simulations will be employed to test this hypothesis. Understanding protein aggregation has implications in all facets of biology as well as on biotechnology and health. At the core of the broader impact activities is training of graduate, undergraduate, and, in particular, high school students. The project is a multidisciplinary study that spans many topics in physics, chemistry, and biology and represents a great platform for further academic endeavors of the involved students. The microscopic hypothesis underlying the proposed work is that the mesoscopic clusters stem from the formation of transient protein-containing complexes. The complexes are stabilized at high protein densities. In contrast with the bulk solution, the complexes are the dominant protein-containing species inside the clusters. In a steady-state cluster, the influx of protein in the form of monomers is exactly balanced by the outflow of protein in the form of complexes. The nature of the complex depends on whether the protein is monomeric, as is lysozyme, or oligomeric, as is hemoglobin, ordinarily a tetrameric protein. The investigators in this study hypothesize that for typically monomeric proteins, complex formation is accompanied by partial protein unfolding and, possibly, domain swapping. In the case of oligomeric proteins, the complexes are oligomers that contain an untypical number of individual monomers. A core aspect of the hypothesis is that the complexes are opportunistic; they represent untypical ways to transiently bind individual protein molecules together. The research team will establish the identity and mechanisms of the complexation using a combination of physicochemical and biochemical experimental techniques (dynamic light scattering, Brownian microscopy, mass spectroscopy, amide exchange NMR, sheer flow) and theoretical tools (molecular modeling employing coarse-grained energy functions and the classical density functional theory). They will test whether the clusters are truly steady-state objects and explore the possibility that cluster formation has a slow, irreversible component. This part of the proposed study may answer the question whether clusters ripen according to an Ostwald-like scenario, or a different mechanism is involved.