To perform their function, proteins must operate in the crowded environment of a living cell, thus requiring mechanisms that prevent protein aggregation. When these mechanisms fail, pathological conditions, such as sickle cell anemia or plaque formation, take place. In some cases, on the other hand, specific types of aggregation are actually desirable; examples including storage of insulin in the pancreas and protein in grains, in the form of crystals. Making protein crystals remains the single most important tool for protein structure determination, which is crucial for understanding protein function. It comes as a surprise that for those protein aggregates to form, folded protein molecules must first organize into long-lived clusters of a protein-rich liquid that are about a micron in size: According to the existing paradigms of phase equilibrium, such mesoscopic clusters should not exist, nor have they been seen in other similar systems such as colloids. The project aims to elucidate the molecular mechanism and the thermodynamic basis of how the puzzling mesoscopic clusters form. To accomplish this goal, the project will combine the theoretical and experimental efforts of the Lubchenko and Vekilov labs using four proteins as model systems: lysozyme, hemoglobin, insulin, and lumazine synthase. The roles of water structuring at the protein-solvent interface and the formation of transient protein oligomers in the stabilization of the protein-rich phase will be investigated by molecular modeling and tested by means of dynamic/static light scattering and thermodynamic and rheological characterizations. The rich kinetics of the formation/decay of clusters resulting from the interplay between protein transport and oligomer formation will be worked out by solving non-linear kinetic schemes coupled with diffusion/advection and tested against measured life-times and sizes of the clusters.
The solution of the important problem of mesoscopic aggregates in concentrated protein solutions lies at the interface of biology, physics, chemistry, and materials science. In addition, this research will be a close collaboration between a theoretical and experimental group from departments of chemistry and chemical engineering. These factors will combine to create a unique multidisciplinary research environment for participating students and research infrastructure. Considering the ethnic diversity at the University of Houston and in the greater Houston area, the research will enhance the educational opportunities in several underrepresented groups and promote their participation in advanced research. Existing collaborations with local writers and radio personalities will be utilized to publicize the societal benefits of the research. In addition to the fundamental and clinical significance of the research, its benefits include new potential routs for manufacturing novel materials and improving the nutritional value of crops. This project is jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences and by the Physics of Living Systems program in the Physics Division.
Intellectual Merit: The research supported by this award focused on a puzzling phenomenon that takes place in protein solutions. In solutions of proteins such as lysozyme, hemoglobin, lumazine synthase, and others, one often finds droplets of protein-rich solution that are about a micron across. We have called these droplets mesoscopic clusters. According to modern views on phase transitions, such mesoscopic droplets should not exist: The volume of the sample should either be fully occupied by a single phase, or should be shared by two phases in comparable measure, as in a mixture of water and ice near the freezing point. In contrast, the presence of the clusters is analogous to micro-icebergs floating in water well above freezing, something that cannot possibly happen. Early studies had indicated that because the clusters are denser than the bulk solution, they might serve as an initial step for forming various solid aggregates of proteins: both unwanted, such as sickle cell anemia fibers, and desirable, such as crystals. The growth of the sickle cell anemia fibers from a macroscopic dense phase is shown in Fig.1. These applications represent an important motivation for the project, in addition to its fundamental significance. The clusters are difficult to study in the laboratory because they contain only a small fraction of the total protein in the solution; this fact, combined with the clusters’ mesoscopic size, had in the past limited our experimental tools to dynamic light scattering. This methodological challenge was also part of our motivation. The research, which is an ongoing collaboration between a theory (Lubchenko) and experimental (Vekilov) teams, has made significant progress toward both fundamental understanding and characterization of the mesoscopic clusters and their underlying causes, and the implications of the clusters for a broad class of protein aggregation phenomena. We have discovered that the clusters form via a novel physicochemical mechanism. The clusters consist of a strongly non-equilibrium mixture of single protein molecules and long-lived complexes of proteins, possibly involving other solutes. The puzzling mesoscopic size originates from a new time scale in the problem, i.e., the lifetime of the complexes. Several important results have been obtained with regard to the microscopic nature of the protein complexes. We have established that in the case of protein lysozyme, which is found in tears or chicken egg whites, the formation of protein complexes must be accompanied by partial unfolding of individual molecules. During our experimental studies, we have discovered that the clusters mature with time similarly to how solid solutions ripen, namely, that the grain size grows with time proportionally to the cubic root of time. Dynamic light scattering data demonstrating the cluster growth are shown in Fig. 2. Despite this similarity between the two systems, the cluster growth is significantly slower, overall, and thus must occur according to a distinct mechanism. This mechanism is currently under investigation. We have developed a new way to observe clusters that is more direct than the dynamic light scattering and allows one to follow individual clusters in real time. This method, which we have called the Brownian Microscopy, exploits a higher fluorescence intensity of clusters compared to the bulk solution. A distinct set of findings concerns the growth of fibers in hemoglobin solutions, of direct significance to research and treatment of sickle cell anemia. We have shown that the fibers indeed nucleate within the mesoscopic clusters, even though the clusters contain only a tiny fraction of the protein. Furthermore we have seen that free heme, which is spontaneously released by hemoglobin, strongly affects the formation of clusters; this is subject of current studies. Broader Impact: The solution of the important problem of mesoscopic aggregates in concentrated protein solutions lies at the interface of biology, physics, chemistry, and materials science. In addition, this research has been a close collaboration between a theoretical and experimental group from departments of chemistry and chemical engineering. These factors combined to create a unique multidisciplinary research environment for participating students and research infrastructure. The research has enhanced the educational opportunities in several underrepresented groups and promoted their participation in advanced research. Four graduate students and two postdoctoral fellows contributed to the research. Seven undergraduate and three high school students have been trained in various aspects of protein science and are now pursuing further studies at premier educational institutions in the US. The research resulted in more than twenty publications and has been publicized at numerous conferences, including interdisciplinary symposia attended by biologists, physicists, chemists, and materials scientists. In addition to the fundamental and clinical significance of the research, its benefits include new potential routs for manufacturing novel materials and improving the nutritional value of crops.
