Intellectual Merit - Non-native aggregation is a ubiquitous hurdle to successful over-expression and purification of recombinant proteins throughout the biotechnology and biopharmaceutical industries. This is particularly the case for multi-domain proteins, such as natural and designed antibodies, as well as for other predominantly-beta proteins. Fundamentally, rational design of these more complex proteins is handicapped by limitations in the mechanistic understanding of key features of sequence and structure that control aggregation of multi-domain proteins.
This project seeks to lay a foundation both to fill the gaps in fundamental understanding of the mechanism(s) of multi-domain protein aggregation, and to provide first-generation design rules to imbue aggregation resistance. Human gamma-D Crystallin (gamma D-Crys) will be the model multidomain protein, chosen because it possesses a number of desirable features: (1) it is a single chain, two domain protein; (2) it represents an important set of all-beta proteins; (3) it has established aggregation and folding behavior; (4) it has available, aggregation-prone mutants.
Broader Impact - The proposed research will result in an improved mechanistic understanding of aggregation and rational design of aggregation resistance for multi-domain, all-beta Greek-key proteins, of which a number of antibody constructs are a subclass of widespread biological and biotechnological interest. The research will also provide a framework for the education and training of graduate and undergraduate students in the PIs' laboratories, with a specific focus on cutting-edge experimental and modeling tools. As in the past, the PIs will involve students drawn from underrepresented groups in science and engineering. Finally, a set of examples and problems will be developed to incorporate aspects of this research into the undergraduate curricula, including computational and modeling activities. This module will be disseminated to other faculty via a web-based repository of educational materials developed at San Jose State University.
The focus of this project was on developing a series of "rules" or "guidelines" that help researchers re-design existing proteins or design new proteins so that are more resilient to heating and other "stresses" that they encounter when one tries to use them as the active drug component of pharmaceuticals. These stresses typically occur during manufacturing and storage of the final product, and the proteins react to this stress by changes their shape and by misassociationwith one another. The resulting aggregates and otherwise distorted proteins are problems because the shape of the molecules are critical for their function as drug molecules, and because the body’s immune system can incorrectly react to these distorted drug proteins as foreign invaders, and activate an immune response. This project considered a number of different molecular-scale modeling approaches, aided by modern high-speed computers, to make predictions about how to change the chemistry of a given protein so as to make it resistive to shape distortions and/or resistive to allowing another protein to agglomerate with it. This is a challenge because the same molecular-scale forces that help a protein maintain its proper shape (i.e., the interactions between pieces of the same protein) are those than allow it to find favorable interactions between proteins, and those interactions between proteins drive agglomeration. As a result, there is a balance between the "internal" interactions within a protein and "external" interactions between a protein molecule and other proteins in solution. The major result of this project from a conceptual perspective was that making the proteins more resistive to interactions between proteins was more effective than trying to keep the protein "folded" into its proper shape. This is rather surprising when one compares to what has been found repeatedly over the past ten to twenty years in numerous examples of how the balance between these "internal" and "external" interactions leads to changes in protein agglomeration. In most of the previous examples, when one changes environmental factors like temperature or the acidity / basicity of the solution, or adds salts or sugars to the system that affect the "internal" and "external" interactions for proteins, the majority of the time the "internal" interactions that affect the structural distortions of the proteins appears to be the dominant factor. This teaches us that the way in which we "design" a protein to resist distortion and agglomeration can be fundamentally different from how we "design" the environment a protein is manufactured or stored in before it reaches a patient. In practice, this can have a large impact on how industrial researchers approach the problem of bringing drug candidates to the market more quickly, with lower costs and ultimate price for the patient or insurers, and will lower risk for adverse side effects in the clinical trials and after the drug is marketed to patients. In addition to the conceptual outcomes, the benefits to broader scientific community and society include: (i) improved computer programs for allowing others to test these concepts with a range of different proteins that may have applications in various industries (e.g., enzymes in washing detergents, medical imaging reagents, enzymes in waste remediation, and new classes of drug molecules); (ii) numerous students were trained via the work that was funded by this project, including women and underrepresented minorities, some of whom are already now in U.S.-based pharmaceutical laboratories, and others are headed there or are in graduate school to help become the next generation of technological leaders in the biotechnology industry.
