Structural biology by magic angle spinning NMR: atomic-resolution structure of cofilin assembled with filamentous actin, and the power of integrated NMR and QM/MM calculations in microcrystalline galectin
Date
2021
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Publisher
University of Delaware
Abstract
The first structure of a protein solved by magic angle spinning (MAS) solid-state NMR spectroscopy, the SH3 domain of a-spectrin, was reported in 2002. Since then, MAS NMR has become a structural biology technique, as it allows for the determination of atomic-resolution structure and site-specific dynamics in large biological systems that are insoluble and cannot be crystallized, including proteins and protein assemblies, biological machines, as well as intact cells. Despite the rapid advances in MAS NMR hardware and pulse sequences, the technique is not yet mature and requires the development of new methods, which is an area of active research in multiple laboratories including ours. ☐ In this thesis, I first examine the requirements for the calculation of accurate 13C and 15N NMR chemical shift tensors in a microcrystalline protein, the carbohydrate-binding domain of galectin-3C. by hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, and using the experimental MAS NMR data and high resolution X-ray crystal structures. The objective of this study is to determine the limits of the accuracy of these calculations with the current state-of-the art Density Functional Theory (DFT). The results of this study show that 13Cα chemical shifts exhibit the highest dependence on local geometry, and can be calculated to the accuracy of within ±2 ppm. The accuracy of the calculated 15NH chemical shifts is much lower, ±6 ppm, and the best agreement with experiment is reached when QM/MM are performed in combination with molecular dynamics (MD) simulations in an explicit solvent environment. Overall, the current results, while promising, underscore the limitations of the current state of the art in QM/MM calculations. ☐ In Chapter 6 of this thesis, I report the atomic-resolution structure of the actin-binding protein, human cofilin-2, in complex with filamentous actin, determined by MAS NMR. The actin depolymerizing factor (ADF)/cofilin family of proteins are potent regulators of actin severing and filament disassembly. However, the structural basis for isoform-specific severing activity is poorly understood due to a lack of high-resolution structures in complex with F-actin. The MAS NMR structure of cofilin-2/actin complex reveals a novel isoform-specific conformation that is a result of a unique network of hydrogen bonding interactions in a non-conserved region of the protein. Furthermore, the results reveal novel binding site interactions, and provide insight for isoform-specific actin filament disassembly. ☐ In Chapter 7 of the thesis, I describe my contributions to the development of 19F MAS NMR spectroscopy for structural analysis of organic and biological molecules, in collaboration with other Polenova group members. 19F offers many advantages in NMR spectroscopy due to its 100% natural abundance, high sensitivity, and low background in biological samples. I first discuss the determination of 19F chemical shift tensors in fluorinated tryptophan compounds. The study reveals that 19F chemical shifts are very sensitive to local environment including the position of fluoro-substitution on the indole ring of the tryptophan sidechain, the solvation state, and the 19F contacts in the crystal lattice. Then, I discuss the determination of interfluorine distance networks using crystalline 7-fluoro-L-tryptophan as an example. The interfluorine distances on the order of 20 Å can be determined on the basis of exchange curves, and this methodology can be directly applied to fluorine distance networks in large protein assemblies with unknown structures.
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Keywords
Nuclear Magnetic Resonance, Magic Angle Spinning, Quantum Mechanics, Molecular Mechanics, Molecular Dynamics