Structural Investigations of Molecular Machines by Solid-State NMR

Abstract

Essential biological processes such as cell motion, signaling,protein synthesis, and pathogen-host interactions rely on multifunctional molecular machines containing supramolecular assemblies, that is, noncovalently assembled protein subunits. Scientists would like to acquire a detailed atomic view of the complete molecular machine to understand its assembly process and functions. Structural biologists have used various approaches to obtain structural information such as X-ray crystallography, solution NMR, and electron microscopy. The inherent insolubility and large size of these multicomponent assemblies restrict the use of solution NMR, and their noncrystallinity and elongated shapes present obstacles to X-ray crystallography studies. Not limited by molecular weight or crystallinity, solid-state NMR (ssNMR) allows for structural investigations of supramolecular assemblies such as helical filaments, cross-β fibrils, or membrane-embedded oligomeric proteins. In this Account, we describe recent progress in the application of ssNMR to the elucidation of atomic structures of supramolecular assemblies. We highlight ssNMR methods to determine the subunit interfaces in symmetric arrangements. Our use of [1-(13)C]- or [2-(13)C]-glucose as a carbon source during bacterial protein expression results in significant (13)C spin dilution that drastically improves the spectral quality and enables us to detect meaningful structural restraints. Moreover, we can unequivocally determine intermolecular restraints using mixed [(1:1)1-(13)C/2-(13)C]-glucose labeled assemblies. We recently illustrated the power of this methodology with the structure determination of the type III secretion system (T3SS) needle. One crucial aspect in elucidating the atomic structure of these large multicomponent complexes is to determine the subunit-subunit interfaces. Notably, we could probe the needle subunit interfaces by collecting (13)C-(13)C intermolecular restraints. In contrast, these interfaces are not accessible via high-resolution cryo-EM. This approach is readily applicable to other supramolecular assemblies containing symmetrically repeating protein subunits, and could be combined with other techniques to get a more complete picture of multicomponent structures. To determine near-atomic structures of assemblies of biological interest, researchers could combine ssNMR data collected at the subunit interfaces with the envelope obtained from cryo-EM and potentially with monomeric subunit crystal structures.

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR

“…It should be emphasized that relative alignment of two AP205CP monomers could be determined from the NMR data without the need for mixed isotope labeling that is often applied to filter intramolecular contacts (67). This is particularly important because there is no protocol for disassembly and reassembly of the AP205CP complex that would be needed for such an approach.…”

Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag

“…Over the past decade magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) has emerged as an invaluable spectroscopic technique capable of yielding atomic level structural and dynamic insights for a wide range of biological macromolecules (McDermott, 2009; Tycko, 2011; Loquet et al, 2013; Yan et al, 2013). Among the most recent additions to the biomolecular solid-state NMR toolbox is a class of methods based on the use of intrinsic or extrinsic paramagnetic probes including organic radicals, transition metal ions and lanthanides (Jaroniec, 2012; Bhaumik et al, 2013; Knight et al, 2013; Parthasarathy et al, 2013; Sengupta et al, 2013).…”

Biosilica and bioinspired silica studied by solid-state NMR

“…[98][99][100][101][102][103][104] The physical condition of a sample is a key determinant for NMR resolution, with crystalline preparation usually yielding more resolved spectra [105]. However, the most interesting targets for solid state NMR are precisely those that are harder (or impossible) to obtain in crystalline form, such as membrane proteins, [5][6][7][106][107][108][109] protein assemblies [110][111][112][113][114], pathological aggregates [57][58][59][60] and fibrils [119][120][121][122][123]3,[124][125][126].…”