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Title:State-of-the-art REDOR and TEDOR methods for elucidation of protein structure and interactions
Author(s):Ghosh, Manali
Director of Research:Rienstra, Chad M.
Doctoral Committee Chair(s):Rienstra, Chad M.
Doctoral Committee Member(s):Chan, Jefferson; Gruebele, Martin H.W.; Tajkhorshid, Emad
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Protein Structure
Abstract:Protein structure determination is vital for elucidating their function, folding or misfolding pathways, and their interaction with other biomolecules, small molecules and membranes. In the field of neurodegenerative disorders like Parkinson’s Disease and Alzheimer’s Disease, amyloid fibrils that constitute the aggregates in the diseased brain are formed due to misfolding of proteins like a-synuclein (a-syn) and amyloid-b. Understanding the structure of these amyloid fibrils will be beneficial for probing amyloid- ligand interactions, amyloid-membrane interactions and even their misfolding pathways. This dissertation thesis introduces some state-of-the-art solid-state nuclear magnetic resonance (SSNMR) techniques to investigate protein structures and their interactions, which will facilitate the study of amyloid fibrils formed in the neurodegenerative disorders. One of the major biophysical techniques to probe biomolecular structure and their interactions is magic angle spinning (MAS) SSNMR spectroscopy due to its ability to examine insoluble systems and with no inherent restriction on the size of the molecule. For structure determination and refinement, we employ famous and robust techniques like rotational echo double resonance (REDOR) and transferred echo double resonance (TEDOR) that recouples the dipolar couplings between two selective heteronuclear spins present in the biomolecule. The dipolar coupling is directly proportional to the gyromagnetic ratio (g) of the spins and indirectly proportional to the cube of the distance between them. Hence, fitting the dipolar dephasing curves to Bessel function of first kind provide us quantitative, precise and unambiguous distance restraints within or between molecules and these distance restraints will assist the structure calculations or docking of small molecules or other biomolecules to the proteins. Structure or ligand binding mode determination with REDOR or TEDOR has been performed extensively on all types of biomolecules, small molecules and inorganic compounds in the past couple of decades. However, due to utilization of lower g nuclei in the spin pair and restrictions in the pulse sequence, the sensitivity, resolution, the distance detection range, and the types of sample on which REDOR and TEDOR can be applied have been limited. In my thesis, I have focused on advancing REDOR and TEDOR methodology such that we can obtain quantitative and precise distances up to 1.5-2 nm. These are the longest quantitative distances obtained by SSNMR so far. This has been made possible by utilizing high g spins, like 1H and 19F as REDOR or TEDOR spins. The novel methods are multidimensional in nature, can be applied to uniformly 13C or 2H labeled proteins, highly sensitive and will be performed in reduced experimental time due to 1H-detection. 1H-detection is feasible under fast MAS and in perdeuterated proteins. Perdeuteration of protein leads to enhanced 1H spin-spin relaxation time (T2), which is a huge advantage for performing REDOR dephasing or TEDOR build-up for longer times and hence increasing the distance detection range. The REDOR or TEDOR techniques introduced in this thesis have been mainly applied on uniformly-13C, 2H, 15N (U-CDN) labeled proteins, like GB1 crystalline protein and a-syn fibrils and their mutants, back-exchanged to 10-30% 1H. 1H-detected REDOR or TEDOR techniques developed here are unique in measuring distances between the side chain 13C atoms and backbone 1H atoms in the proteins, thereby providing accurate restraints for side chain orientations and not only backbone restraints. Performing REDOR with 19F atom is advantageous because of the usefulness and popularity of 19F atom in the drug or diagnostic agent development industry. In our studies, we have also successfully demonstrated the incorporation of 19F atom in a-syn fibrils through mutagenesis and chemical modification. Thereafter, we performed 13C-19F REDOR to obtain distances of up to 10 Å and with our newly developed 1H-detected REDOR pulse sequence, we obtained 1H-19F unambiguous REDOR distances of up to 17 Å in a-syn fibrils. Taken together, this thesis lays a foundation towards shaping REDOR and TEDOR spectroscopy to be feasible for — (1) structure determination and refinement of uniformly 13C labeled and perdeuterated proteins, and (2) binding mode determination of fluorinated imaging agents or drugs, and lipid membranes to amyloid fibrils like a-syn, or non-fluorinated small molecules to 19F- labeled proteins.
Issue Date:2019-06-18
Rights Information:Copyright 2019 Manali Ghosh
Date Available in IDEALS:2019-11-26
Date Deposited:2019-08

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