Unraveling the nanoscale processes of biological pathways via the testing, replication, and visualization of the underlying mechanisms remains a persistent challenge in the study of these critical life-governing systems. Recent advances in the field of chemically induced dimerization have unlocked multiple tools for the exploration of these facets of biology, including the development of switchable signaling systems, assertion of control over protein localization in the cell, and regulation of gene expression. An additional revelation through protein complexation by chemical induction is the construction of multivalent protein-based nanostructures, capable of bearing multiple targeting agents. However, stochastic assembly of these proteins has proven unsatisfactory in generating homogeneous populations. Herein, we have taken the initial steps toward developing a protein-based biomolecular language for nanostructural assembly. Through gel filtration analysis, we have characterized the ability of interfacial point mutations to modulate the stability of a bis-methotrexate (bis-MTX) induced E. coli dihydrofolate reductase (DHFR) dimer over a dynamic range of 1.5 kcal/mol. Furthermore, we have employed single-molecule fluorescence assays to demonstrate the stabilization of a heterodimeric DHFR dimer, yielding 4-fold selectivity for the heterodimer over either corresponding homodimer.
In addition to our experimental characterization of the chemically induced DHFR dimer, we have also taken steps toward the construction of a tripartite computational model of dimerization in an effort to predict the effects of further mutations. We have tested a number of molecular mechanics force fields against quantum mechanical benchmarks and discovered that the MMFF94, OPLS2005, and AMBER force fields yield the most accurate electrostatic and configurational treatment of the complex bis-MTX dimerizer. While initial attempts at calculating the binding free energy of the macromolecular complex have been unsuccessful, we have gleaned important insights into the complexities of modeling this three-body system. The advances described within the following work delineate important aspects of protein interface remodeling in a chemically induced system and provide an avenue toward the further development of both a computational model of protein interactions and the future directed assembly of protein based materials and therapeutic nanostructures.
University of Minnesota Ph.D. dissertation. November 2009. Major: Medicinal Chemistry. Advisor: Dr. Carston R. Wagner. 1 computer file (PDF); xv, 252 pages, appendices I-III. Ill. (some col.)
White, Brian Richard.
Toward therapeutic nanoassemblies: the design and modeling of protein-protein interactions..
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