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• Title: High-Resolution Structural Studies of Paramagnetic Proteins by Multidimensional Solid-State Nuclear Magnetic Resonance Spectroscopy
• Author : Philippe S. Nadaud
• Release Date : January 19, 2013
• Genre: Medical,Books,Professional & Technical,
• Pages : * pages
• Size : 15115 KB

Description

Nuclear magnetic resonance (NMR) is one of the major spectroscopic techniques available for the characterization of molecular structure and conformational dynamics with atomic level detail. NMR relies on the intrinsic magnetic properties of certain nuclear isotopes, such as 1H, 13C, 15N, and 31P, which provide convenient, site-specific structural probes when placed inside a large external magnetic field. Recent developments in solid-state NMR (SSNMR) spectroscopy promise to enable detailed structural studies to be performed for important biological macromolecules, which are inherently insoluble but at the same time cannot be readily crystallized for analysis by X-ray diffraction. Prominent examples of systems of this type include large macromolecular complexes, membrane-bound peptides and proteins important in cell signaling, and fibrillar protein aggregates associated with the development of systemic and neurodegenerative human disorders, including Alzheimer’s and Parkinson’s diseases and type II diabetes.One of the main outstanding problems in biomolecular solid-state NMR spectroscopy, which impedes the widespread structural studies of proteins using this methodology, is related to the paucity of long range (i.e., greater than 5 A) structural restraints that can be determined by the currently available SSNMR techniques. This is primarily due to the fact that through space magnetic dipole-dipole interactions between 1H, 13C, 15N nuclei, which report on interatomic distances and thus protein structure, become vanishingly small for distances significantly in excess of ~5-6 A. The underlying fundamental concept of the new SSNMR methods described in this thesis is that, when introduced into normally diamagnetic proteins, unpaired electrons, which like certain nuclei are also magnetic particles, can couple very strongly through space to the neighboring nuclei. Indeed these electron-nucleus interactions are several orders of magnitudes larger than typical internuclear magnetic dipole-dipole couplings due to the large magnetic moment of the electron spin. One well-known physical phenomenon, which is a direct consequence of these large electron-nucleus interactions is the so-called nuclear paramagnetic relaxation enhancement (PRE). Importantly, the PRE phenomenon depends on the electron-nucleus separation in a well-defined manner and can extend to distances of up to ~15-20 A.The observation and quantification of long-range PRE phenomena in the solid phase has great potential for investigation of protein structures by SSNMR spectroscopy. However, no SSNMR study to date has experimentally demonstrated the systematic measurement of PRE effects in uniformly 13C,15N isotope labeled proteins. To address this we have designed a series of SSNMR experiments for the detection and quantification of nuclear PREs in model protein molecules intentionally modified with covalently-attached non-native sidechains containing various paramagnetic centers including nitroxide spin labels and transition metal ions. Most significantly, in the case of proteins containing Cu2+ ions, in the form of EDTA-chelates, we were able to demonstrate the quantitative measurements of electron-nucleus distances up to ~20 A. Both the quality and the information content of these PRE-based structural restraints suggest that they will become useful in future applications of SSNMR spectroscopy to the rapid refinement of three-dimensional protein folds.

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