The functions of most cellular proteins are understood to depend on their shape or structure. Many proteins, however, include parts where the structure is not well defined. The shape and thus the functions of these intrinsically disordered regions (IDRs) of proteins are difficult study. With this award, the Chemistry of Life Processes Program in the Chemistry Division is supporting the research of Dr. John Antos and Dr. Sergey Smirnov from Western Washington University to develop experimental methods to study the structures of IDRs. The research team is using use a splicing enzyme (sortase) to insert specific chemical tags into proteins that allow their shapes and changes to their shapes to be monitored by Nuclear Magnetic Resonance (NMR) spectroscopy. This project applies these methods to determine the key functional properties of dematin, a protein with a large IRD that helps control red blood cell shape and function. Results from these studies set the stage to study many types of IDRs in proteins that are prevalent across multiple kingdoms of life. The project prepares the next generation of scientists by training participating undergraduate and Masters level graduate students in state-of-the-art methods for analyzing protein structure and function. The project provides research opportunities to engage and attract underrepresented groups and high school students into the natural sciences. In addition, Drs. Antos and Smirnov incorporate authentic research experiences into Course-based Undergraduate Research Experiences (CUREs) for biochemistry courses.
Approximately one-third of eukaryotic proteins contain within their polypeptide sequences sizable intrinsically disordered regions (IDRs), which are critical for a variety of cellular functions. Many of these IDRs are large (hundreds of residues), which makes the site-specific study of these dynamic protein segments and their binding interfaces technically challenging. For example, the application of NMR, one of the preferred methods for studying IDRs, is hampered by severe spectral overlap of resonances originating from large IDRs. Similarly, the targeted insertion of functional labels such as spectroscopic reporters or post-translational modifications is often impossible due to competing labeling of multiple identical sites in extended IDR sequences. This project addresses such problems via robust and efficient site-specific sortase-mediated ligation strategies tuned for large IDR-containing substrates. This includes the development of guidelines for the use of site-specific ligation strategies in the context of large IDR-containing protein substrates. The set of methods developed lead to the generation of derivatives of the erythrocyte-controlling protein dematin segmentally labeled with NMR-active isotopes (15N and 13C). Such derivatives enable characterization of a crucial regulatory intramolecular binding interface involving the large, 315-residue IDR that is central for dematin function and regulation.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.