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Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy.

ABSTRACT: Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of interactions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but so far has been precluded by the low tubulin yield. We present a protocol to produce [13C, 15N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies. This approach allowed us to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. Our results pave the way for in-depth NMR studies of protein dynamics involved in MT assembly and their interactions with other cellular components.

SUBMITTER: Luo Y

PROVIDER: S-EPMC6940360 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy.

Luo Yanzhang Y Xiang ShengQi S Hooikaas Peter Jan PJ van Bezouwen Laura L Jijumon A S AS Janke Carsten C Förster Friedrich F Akhmanova Anna A Baldus Marc M

Nature communications 20200102 1

Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of interactions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but s ...[more]

PMID: 31896752

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Project description:Quadrupolar solid-state NMR carries a wealth of structural information, including insights about chemical environments arising through the determination of local coupling parameters. Current methods can successfully resolve these parameters for individual sites using sample-spinning methods techniques applicable to quadrupolar I ≥ 1 nuclei, provided second-order central transition broadenings do not exceed by much the spinning rate. For large quadrupolar coupling (C Q) values, however, static acquisitions are often preferable, leading to challenges in extracting local structural information. This study explores the use of two-dimensional QUadrupolar Isotope Correlation SpectroscopY (QUICSY) experiments as a means to increase the NMR spectral resolution and enrich the characterization of quadrupolar NMR patterns under static conditions. QUICSY seeks to correlate the solid-state NMR powder line shapes for two quadrupolar isotopes belonging to the same element via a 2D experiment. In general, two isotopes of the same element will have different nuclear quadrupole moments, gyromagnetic ratios, and spin numbers but essentially identical chemical environments. The possibility then arises of obtaining sharp "ridges" in these 2D correlations, even in static samples showing large quadrupolar effects, which lead to second-order line shapes that are several kilohertz wide. Moreover, pairs of quadrupolar isotopes are recurrent in the periodic table and include important elements such as 35,37Cl, 69,71Ga, 79,81Br, and 85,87Rb. The potential of this approach is explored theoretically and experimentally on two rubidium-containing salts: RbClO4 and Rb2SO4. We find that each compound gives rise to distinctive 2D QUICSY line shapes, depending on the quadrupolar and chemical shift anisotropy (CSA) parameters of its sites. These experimental line shapes show good agreement with analytically derived 2D spectra relying on literature values of the quadrupolar and CSA tensors of these compounds. The approach underlined here paves the way toward better characterization of wideline NMR spectra of quadrupolar nuclei possessing different nuclear isotopes.

Dataset Information

Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy.

Publications

Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy.

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