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Resolution of angstrom-scale protein conformational changes by analyzing fluorescence anisotropy.

ABSTRACT: Conformational changes within typical protein molecules are rapid and small, making their quantitative resolution challenging. These changes generally involve rotational motions and may thus be resolved by determining changes in the orientation of a fluorescent label that assumes a unique orientation in each conformation. Here, by analyzing fluorescence intensities collected using a polarization microscope at a rate of 50 frames per second, we follow the changes of 10-16° in the orientation of a single bifunctional rhodamine molecule attached to a regulator of conductance to K+ (RCK) domain of the MthK channel, and thus, the transitions between its three conformational states, with effective standard deviation (?) of 2-5°. Based on available crystal structures, the position of the fluorophore's center differs by 3.4-8.1?Å among the states. Thus, the present approach allows the resolution of protein conformational changes involving ångström-scale displacements.

SUBMITTER: Lewis JH

PROVIDER: S-EPMC6822697 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Resolution of ångström-scale protein conformational changes by analyzing fluorescence anisotropy.

Lewis John H JH Lu Zhe Z

Nature structural & molecular biology 20190905 9

Conformational changes within typical protein molecules are rapid and small, making their quantitative resolution challenging. These changes generally involve rotational motions and may thus be resolved by determining changes in the orientation of a fluorescent label that assumes a unique orientation in each conformation. Here, by analyzing fluorescence intensities collected using a polarization microscope at a rate of 50 frames per second, we follow the changes of 10-16° in the orientation of a ...[more]

PMID: 31488909

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Project description:Resists that enable high-throughput and high-resolution patterning are essential in driving the semiconductor technology forward. The ultimate patterning performance of a resist in lithography is limited because of the trade-off between resolution, line-width roughness, and sensitivity; improving one or two of these parameters typically leads to a loss in the third. As the patterned feature sizes approach angstrom scale, the trade-off between these three metrics becomes increasingly hard to resolve and calls for a fundamental rethinking of the resist chemistry. Low-molecular-mass monodispersed metal-containing resists of high atom economy can provide not only very high resolution but also very low line-width roughness without sacrificing sensitivity. Here we describe a modular metal-containing resist platform (molecular mass <500 Da) where a molecular resist consists of just two components: a metal and a radical initiator bonded to it. This simple system not only is amenable to high-resolution electron beam lithography (EBL) and extreme ultraviolet lithography (EUVL) but also unites them mechanistically, giving a consolidated perspective of molecular and chemical processes happening during exposure. Irradiation of the resist leads to the production of secondary electrons that generate radicals in the initiator bonded to metal. This brings about an intramolecular rearrangement and causes solubility switch in the exposed resist. We demonstrate record 1.9-2.0 nm isolated patterns and 7 nm half-pitch dense line-space features over a large area using EBL. With EUVL, 12 nm half-pitch line-space features are shown at a dose of 68 mJ/cm2. In both of these patterning techniques, the line-width roughness was found to be ≤2 nm, a record low value for any resist platform, also leading to a low-performance trade-off metric, Z factor, of 0.6 × 10-8 mJ·nm3. With the ultimate resolution limited by instrumental factors, potential patterning at the level of a unit cell can be envisaged, making low-molecular-mass resists best poised for angstrom-scale lithography.

Dataset Information

Resolution of angstrom-scale protein conformational changes by analyzing fluorescence anisotropy.

Publications

Resolution of ångström-scale protein conformational changes by analyzing fluorescence anisotropy.

Similar Datasets

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