Multidimensional magic-angle spinning solid-state nuclear magnetic resonance (SSNMR) methods can now be applied to uniformly 13C,15N enriched proteins, enabling detailed atomic-level studies and even the de novo determination of three-dimensional molecular structure. However, one of the outstanding problems in the field, which impedes these efforts, is related to the paucity of long range (i.e., greater than 5 A) distance restraints that can be obtained using the existing SSNMR techniques. In this CAREER project, proteins modified with covalently-bound nitroxide spin labels and paramagnetic metal ions, will be investigated using solid-state NMR, with the main goal of deriving structural restraints on length scales inaccessible to traditional SSNMR methods (up to ~20 A). The specific research objectives are: (i) to explore in detail the paramagnetic relaxation enhancement (PRE) of nuclear spins in the solid phase caused by the dipole-dipole coupling to unpaired electrons, and (ii) to establish novel PRE-based SSNMR methods and apply them to long-range distance measurements, studies of protein-protein interactions, and systematic simplification of complex NMR spectra in challenging biological solids. The new PRE-based SSNMR methods can be adapted to a wide variety of problems involving molecular structure and interactions in non-crystalline biological systems.
The main objective of the education-outreach activities is to directly impact students in the early stage of their educational experience, particularly students from groups traditionally underrepresented in science, technology, engineering and mathematics (STEM) by introducing them to current, interdisciplinary science at the interface of chemistry, biology, and physics and encouraging them to think of science in a broad, discovery-based manner, with the ultimate goal of increasing student participation, retention and graduation rates in the STEM fields. The specific education-outreach activities include: (i) a summer research internship program with a team of undergraduate and local high-school students, (ii) an undergraduate laboratory module, which will introduce modern NMR spectroscopy to second-year undergraduates and will interface with other research-oriented laboratory modules, and (iii) a seminar course for a diverse audience of first-year undergraduate students directly related to the PI's research interests in protein structure and function. This project is jointly supported by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Experimental Physical Chemistry Program in the Division of Chemistry in the Mathematical and Physical Sciences Directorate.
Many large protein complexes that play key roles in fundamental biological processes are inaccessible to conventional structural biology techniques, such as single-crystal X-ray diffraction, yet make suitable targets for in depth structural and dynamic analysis by solid-state nuclear magnetic resonance (NMR) spectroscopy. Although the field of biological solid-state NMR has experienced tremendous growth over the past decade, one ongoing challenge for the elucidation of three-dimensional (3D) protein structures via conventional solid-state NMR approaches is the relative lack of critical data reporting on long-range internuclear distances. During the course of this NSF CAREER project we have introduced new solid-state NMR methodology for the rapid determination of 3D protein structures based on the tagging of protein molecules with paramagnetic centers and measurements of multiple long-range distances between the unpaired electrons of the paramagnetic tag and the protein nuclei. Importantly, these new methods, which were developed using model globular proteins, can be readily extended to much more complex biomolecules such as fibrillar protein aggregates, membrane-bound proteins and other large supramolecular protein assemblies. The CAREER project has also had a significant broader impact on student training in the chemical and biochemical sciences, by providing direct hands-on research experience in cutting-edge NMR spectroscopy and modern biochemistry and molecular biology techniques to a group of nearly 30 graduate, undergraduate and high-school students. The students were introduced to interdisciplinary science at the interface of chemistry, biology and physics, and exposed to scientific research in a highly collaborative environment with numerous opportunities for the most senior graduate students to serve as mentors to their junior graduate, undergraduate and high-school student colleagues.
