Advanced Solid-State NMR Approaches for Structure Determination of Membrane Proteins and Amyloid Fibrils

Department of Chemistry, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.
Accounts of Chemical Research (Impact Factor: 22.32). 05/2013; DOI: 10.1021/ar4000168
Source: PubMed


Solid-state NMR (SSNMR) spectroscopy has become an important technique for studying the biophysics and structure biology of proteins. This technique is especially useful for insoluble membrane proteins and amyloid fibrils, which are essential for biological functions and are associated with human diseases. In the past few years, as major contributors to the rapidly advancing discipline of biological SSNMR, we have developed a family of methods for high-resolution structure determination of microcrystalline, fibrous, and membrane proteins. Key developments include order-of-magnitude improvements in sensitivity, resolution, instrument stability, and sample longevity under data collection conditions. These technical advances now enable us to apply new types of 3D and 4D experiments to collect atomic-resolution structural restraints in a site-resolved manner, such as vector angles, chemical shift tensors, and internuclear distances, throughout large proteins. In this Account, we present the technological advances in SSNMR approaches towards protein structure determination. We also describe the application of those methods for large membrane proteins and amyloid fibrils. Particularly, the SSNMR measurements of an integral membrane protein DsbB support the formation of a charge-transfer complex between DsbB and ubiquinone during the disulfide bond transfer pathways. The high-resolution structure of the DsbA-DsbB complex demonstrates that the joint calculation of X-ray and SSNMR restraints for membrane proteins with low-resolution crystal structure is generally applicable. The SSNMR investigations of α-synuclein fibrils from both wild type and familial mutants reveal that the structured regions of α-synuclein fibrils include the early-onset Parkinson's disease mutation sites. These results pave the way to understanding the mechanism of fibrillation in Parkinson's disease.

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    • "The theoretical and experimental developments of the last years have brought protein solid state NMR (SSNMR) in direct competition with state of the art solution NMR. Furthermore, there is a large number of samples that can be studied at atomic resolution only by SSNMR such as fibrils [1] [2] [3], membrane proteins [3] [4] [5] [6] and large protein aggregates [7] [8] [9] [10], plus a number of hybrid protein-material samples that greatly benefit from the study of both the protein and the material sides by means of SSNMR [11] [12] [13] [14] [15]. "
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    ABSTRACT: Resolution and sensitivity in solid state NMR (SSNMR) can rival the results achieved by solution NMR, and even outperform them in the case of large systems. However, several factors affect the spectral quality in SSNMR samples, and not all systems turn out to be equally amenable for this methodology. In this review we attempt at analyzing the causes of this variable behavior and at providing hints to increase the chances of experimental success. Copyright © 2015 Elsevier Inc. All rights reserved.
    Preview · Article · Mar 2015 · Journal of Magnetic Resonance
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    • "The variety of methods that currently allow for successful research on such structures in a close to in vivo state is very limited. One of them is solid-state NMR spectroscopy (ssNMR), as it enables to study the structure of solid samples that lack a long-distance order [1]. Often, the sample preparation can be carried out in such a way that the biological system under study resembles the in vivo state very closely, which makes this technique yield meaningful results. "
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    ABSTRACT: Solid-state NMR is a versatile tool to study structure and dynamics of insoluble and non-crystalline biopolymers. Supramolecular protein assemblies are formed by self-association of multiple copies of single small-sized proteins. Because of their high degree of local order, solid-state NMR spectra of such systems exhibit an unusually high level of resolution, rendering them an ideal target for solid-state NMR investigations. Recently, our group has solved the structure of one particular supramolecular assembly, the type-iii-secretion-system needle. The needle subunit comprises around 80 residues. Many interesting supramolecular assemblies with unknown structure have subunits larger in size, which requires development of tailored solid-state NMR strategies to address their structures. In this “Perspective” article, we provide a view on different approaches to enhance sensitivity and resolution in biological solid-state NMR with a focus on the possible application to supramolecular assemblies with large subunit sizes.
    Full-text · Article · Nov 2014 · Journal of Magnetic Resonance
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    • "While magic angle spinning (MAS) solid-state NMR spectroscopy has been highly valuable for the atomic-level characterization of a variety of non-soluble and non-crystalline solids [1] [2] [3], recent technical advances have swiftly expanded its range of applications [4] [5] [6] [7] [8] [9]. In particular, the development of ultrafast MAS techniques has initiated numerous exciting discoveries [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]. "
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    ABSTRACT: The refocused insensitive nuclei enhanced by polarization transfer (RINEPT) technique is commonly used for heteronuclear polarization transfer in solution and solid-state NMR spectroscopy. Suppression of dipolar couplings, either by fast molecular motions in solution or by a combination of MAS and multiple pulse sequences in solids, enables the polarization transfer via scalar couplings. However, the presence of unsuppressed dipolar couplings could alter the functioning of RINEPT, particularly under fast/ultrafast MAS conditions. In this study, we demonstrate, through experiments on rigid solids complemented by numerical simulations, that the polarization transfer efficiency of RINEPT is dependent on the MAS frequency. In addition, we show that heteronuclear dipolar coupling is the dominant factor in the polarization transfer, which is strengthened by the presence of (1)H-(1)H dipolar couplings. In fact, the simultaneous presence of homonuclear and heteronuclear dipolar couplings is the premise for the polarization transfer by RINEPT, whereas the scalar coupling plays an insignificant role under ultrafast MAS conditions on rigid solids. Our results additionally reveal that the polarization transfer efficiency decreases with the increasing duration of RF pulses used in the RINEPT sequence.
    Full-text · Article · Apr 2014 · Journal of Magnetic Resonance
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