Solid-State NMR Spectroscopy of Protein Complexes
ABSTRACT Protein-protein interactions are vital for many biological processes. These interactions often result in the formation of protein assemblies that are large in size, insoluble, and difficult to crystallize, and therefore are challenging to study by structure biology techniques, such as single crystal X-ray diffraction and solution NMR spectroscopy. Solid-state NMR (SSNMR) spectroscopy is emerging as a promising technique for studies of such protein assemblies because it is not limited by molecular size, solubility, or lack of long-range order. In the past several years, we have applied magic angle spinning SSNMR-based methods to study several protein complexes. In this chapter, we discuss the general SSNMR methodologies employed for structural and dynamics analyses of protein complexes with specific examples from our work on thioredoxin reassemblies, HIV-1 capsid protein assemblies, and microtubule-associated protein assemblies. We present protocols for sample preparation and characterization, pulse sequences, SSNMR spectra collection, and data analysis.
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ABSTRACT: The capsid protein (CA) of human immunodeficiency virus 1 (HIV-1) assembles into a cone-like structure that encloses the viral RNA genome. Interestingly, significant heterogeneity in shape and organization of capsids can be observed in mature HIV-1 virions. In vitro, CA also exhibits structural polymorphism and can assemble into various morphologies, such as cones, tubes, and spheres. Many intermolecular contacts that are critical for CA assembly are formed by its C-terminal domain (CTD), a dimerization domain, which was found to adopt different orientations in several X-ray and NMR structures of the CTD dimer and full-length CA proteins. Tyr145 (Y145), residue two in our CTD construct used for NMR structure determination, but not present in the crystallographic constructs, was found to be crucial for infectivity and engaged in numerous interactions at the CTD dimer interface. Here we investigate the origin of CA structural plasticity using solid-state NMR and solution NMR spectroscopy. In the solid state, the hinge region connecting the NTD and CTD is flexible on the millisecond time scale, as evidenced by the backbone motions of Y145 in CA conical assemblies and in two CTD constructs (137-231 and 142-231), allowing the protein to access multiple conformations essential for pleimorphic capsid assemblies. In solution, the CTD dimer exists as two major conformers, whose relative populations differ for the different CTD constructs. In the longer CTD (144-231) construct that contains the hinge region between the NTD and CTD, the populations of the two conformers are likely determined by the protonation state of the E175 side chain that is located at the dimer interface and within hydrogen-bonding distance of the W184 side chain on the other monomer. At pH 6.5, the major conformer exhibits the same dimer interface as full-length CA. In the short CTD (150-231) construct, no pH-dependent conformational shift is observed. These findings suggest that the presence of structural plasticity at the CTD dimer interface permits pleiotropic HIV-1 capsid assembly, resulting in varied capsid morphologies.Journal of the American Chemical Society 03/2012; 134(14):6455-66. DOI:10.1021/ja300937v · 11.44 Impact Factor
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ABSTRACT: Using the DUMAS (Dual acquisition Magic Angle Spinning) solid-state NMR approach, we created new pulse schemes that enable the simultaneous acquisition of three dimensional (3D) experiments on uniformly (13)C, (15)N labeled proteins. These new experiments exploit the simultaneous cross-polarization (SIM-CP) from (1)H to (13)C and (15)N to acquire two 3D experiments simultaneously. This is made possible by bidirectional polarization transfer between (13)C and (15)N and the long living (15)N z-polarization in solid state NMR. To demonstrate the power of this approach, four 3D pulse sequences (NCACX, CANCO, NCOCX, CON(CA)CX) are combined into two pulse sequences (3D DUMAS-NCACX-CANCO, 3D DUMAS-NCOCX-CON(CA)CX) that allow simultaneous acquisition of these experiments, reducing the experimental time by approximately half. Importantly, the 3D DUMAS-NCACX-CANCO experiment alone makes it possible to obtain the majority of the backbone sequential resonance assignments for microcrystalline U-(13)C,(15)N ubiquitin. The DUMAS approach is general and applicable to many 3D experiments, nearly doubling the performance of NMR spectrometers.Journal of Magnetic Resonance 04/2012; 220:79-84. DOI:10.1016/j.jmr.2012.04.006 · 2.32 Impact Factor
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ABSTRACT: We report dramatic sensitivity enhancements in multidimensional MAS NMR spectra by the use of nonuniform sampling (NUS) and introduce maximum entropy interpolation (MINT) processing that assures the linearity between the time and frequency domains of the NUS acquired data sets. A systematic analysis of sensitivity and resolution in 2D and 3D NUS spectra reveals that with NUS, at least 1.5- to 2-fold sensitivity enhancement can be attained in each indirect dimension without compromising the spectral resolution. These enhancements are similar to or higher than those attained by the newest-generation commercial cryogenic probes. We explore the benefits of this NUS/MaxEnt approach in proteins and protein assemblies using 1-73-(U-(13)C,(15)N)/74-108-(U-(15)N) Escherichia coli thioredoxin reassembly. We demonstrate that in thioredoxin reassembly, NUS permits acquisition of high-quality 3D-NCACX spectra, which are inaccessible with conventional sampling due to prohibitively long experiment times. Of critical importance, issues that hinder NUS-based SNR enhancement in 3D-NMR of liquids are mitigated in the study of solid samples in which theoretical enhancements on the order of 3-4 fold are accessible by compounding the NUS-based SNR enhancement of each indirect dimension. NUS/MINT is anticipated to be widely applicable and advantageous for multidimensional heteronuclear MAS NMR spectroscopy of proteins, protein assemblies, and other biological systems.The Journal of Physical Chemistry B 06/2012; 116(25):7416-27. DOI:10.1021/jp3032786 · 3.38 Impact Factor