Anisotropic Nuclear Spin Interactions for the Morphology Analysis of Proteins in Solution by NMR Spectroscopy

Abstract

NMR spectroscopy has become a standard method to determine the high-resolution structure of biomolecules, including protein, nucleic acids and their complexes.Conventional NMR structural determination is based on distance restraints obtained from proton-proton nuclear Overhauser effects (NOEs), which give approximate distances between interacting protons close together in space (< 5 -6 Å), and the torsion angles through vicinal spin couplings. 1 These NMR parameters all give shortrange structural information.Although the observable maximum distance by NOEs is limited, basically less than 5 Å, the short-range structural constraints can connect parts of a molecule that are far apart in the primary sequence, but close together in the three-dimensional coordinate of a globular protein. In this way, the short-range structural constraints successfully allowed accurate determination of secondary and tertiary molecular geometry of globular proteins.1 In spite of the great success of conventional NMR for the structural determination of globular proteins, there are significant limitations in determining multidomain protein structures that possess a hinge region situated between domains. In such cases, the number of proton distances may be insufficient to define the spatial arrangement of the respective domains. Furthermore, the NOE-derived distance restraints have limited accuracy; the parts defined by the sparse NOE interactions are not fully reliable. Thus, the relative positions of distant parts of extended or modular proteins are often poorly defined.In many multidomain proteins, the relative orientation of domains change in response to ligand binding or covalent modification, which includes phosphorylation, acetylation, methylation and so forth. Agency,[3][4][5] Chiyoda, Japan Determining the relative orientation of domains within a protein is an important problem in structural biology, which has been difficult to address by either X-ray crystallography or NMR. The structure of a multidomain protein in a crystal lattice can be altered by crystal packing forces, resulting in different domain arrangements from those in solution. On the other hand, conventional NMR primarily provides short-range structural information, including proton-proton distances derived from nuclear Overhauser effects (NOEs) and torsion angles through vicinal spin couplings. Thus, NMR cannot always determine the precise interdomain arrangements due to the sparsely observed spin interactions between domains. However, the weak alignment of proteins in solution has enabled a new NMR technique to determine the domain arrangement based on the different structural information, which defines the orientation of a structural unit in protein against the magnetic field. This technique relies on the anisotropic nuclear spin interactions that only occur for a molecule in a weakly aligned state. In this review, the basics of the new NMR approach are described with focusing on its application to domain orientation analysis. We also describe our recently est...

Mobility Capillary Electrophoresis-Restrained Modeling Method for Protein Structure Analysis in Mixtures

“…Therefore, great efforts have been made to resolve the structure of proteins. For example, mass spectrometry (MS) and tandem MS-based proteomics methods have been developed to analyze proteins in large scale, and mainly the primary sequence and post-translational modifications (PTMs) could be obtained. − On the other hand, the high-resolution protein stereostructure could be resolved using techniques such as nuclear magnetic resonance (NMR), X-ray crystallography, and cryo-electron microscopy. − These high-resolution techniques typically require high protein concentrations and complicated sample preparation processes, such as labeling and crystallization. − Although MS-based methods could analyze the trace amount of proteins in mixtures, very limited protein stereostructure information could be obtained.…”

Preferential domain orientation of HMGB2 determined by the weak intramolecular interactions mediated by the interdomain linker

“…The relative orientation of the HMG domains in the HMGB2-AlB fragment was estimated based on the alignment tensors obtained for each domain. The alignment induced TROSY shift changes were used to determine the alignment tensor for each HMG domain, which is referred to as DIORITE [5,31]. Because of the "L-shape" of each HMG domain and the entirely extended structure of the HMGB2-AlB fragment, which has two HMG domains, the signals showed severe broadening under weak-alignment conditions; most of the anti-TROSY components on the IPAP-HSQC [32] spectra for the HMGB2-AlB fragment showed poor signals or disappeared.…”

“…The changes in the fluctuation of the interdomain linker by mutations, P80G/P81G and Y78G, should alter the interdomain dynamics in HMGB2-AlB. The interdomain dynamics was elucidated through the alignment tensors determined by the residual pseudo- 15 N CSA (RPCSA), which are measured as the TROSY chemical shift differences between the isotropic and aligned states [4,5,31,38].…”

“…Alternative approaches are available for the structural elucidation of the modular proteins. Another type of NMR experiment using residual dipolar couplings (RDCs) provides long-range structural information that defines the relative domain orientation and interdomain dynamics [2][3][4][5][6][7][8]. Small angle X-ray scattering (SAXS) is also used for the same purpose by its ability to give molecular shape information [9].…”

Complementary Use of NMR to X-Ray Crystallography for the Analysis of Protein Morphological Change in Solution

“…DIORITE analysis, thus, can take full advantage of the TROSY effect on a 900 MHz NMR spectrometer, which is now commercially available. (Tate 2008, Tate et al 2004. In general, chemical shift is very sensitive to the solution conditions, including temperature, sample concentration, pH, ionic strength and so no.…”