New tools derived from the solvatochromic 4-N,N-dimethylamino-1,8-naphthalimide fluorophore for the detection of biomolecular interactions

Abstract

The concept that complex cellular behavior is driven by an intricate network of biomolecular interactions, which emerged through billions of years of evolution, is one of the great wonders of nature. Living cells must constantly assimilate vital information from the surrounding environment and respond appropriately. Such responses include growth, proliferation, migration, and even death. Much of this activity is tightly regulated through elaborately integrated signal transduction pathways - sometimes involving hundreds of cellular components. In order to further our understanding of the basic molecular machinery responsible for coordinating life processes at the cellular level, new tools are required. Fluorescence methods have formed a cornerstone of modern cell biology and have allowed researchers to interrogate the dynamics of many biomolecular events in real-time. However, the criteria for a good fluorescent probe are often numerous and demanding, particularly when considering applications in complex chemical environments like that of a living cell. Tradeoffs often exist between variables such as good extinction coefficients, suitable wavelengths of excitation and emission, thermal and photochemical stability, size, and others. Here in, we report a new series of fluorescent tools based on the solvatochromic 4-N,Ndimethylamino- 1,8-naphthalimide (4-DMN) fluorophore for the study of biomolecular interactions. This fluorophore is distinctive in that it yields very few compromises by combining many of the key elements required of a good fluorescent tool.

(cont.) It can be excited in the visible spectrum (400-450 nm), thus minimizing the damaging effects of high-energy UV light; derivatives of the dye can be prepared in few synthetic steps for facile incorporation into peptides and proteins; it is highly stable to a wide range of chemical conditions; and it possesses extremely sensitive switch-like fluorescent properties capable of responding to subtle changes in the local solvent environment. This thesis chronicles the design and validation of a new fluorescent amino acid of the 4-DMN dye as well as. a series of thiol-modifying agents for application in protein and peptide studies. The power of these new tools is demonstrated using the calcium binding protein calmodulin as a model system. Ongoing work to develop a sensitive sensor of Cdc42 activity is also described.