Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label

Heaven, Graham; Hollas, Michael A.; Tabernero, Lydia; Fielding, Alistair J.

doi:10.1007/s00723-021-01350-1

Cited by 2 publications

(1 citation statement)

References 47 publications

(63 reference statements)

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“…An additional advantage of R9 is the thioether linkage that is not cleaved by the commonly employed reducing agents. Other spin label side chains linked to the protein via a thioether have been employed [ 81 , 82 , 96 – 101 ], but have not yet been characterized at the level required for interpreting EPR spectra in terms of protein structure and dynamics.…”

Section: Discussionmentioning

confidence: 99%

A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins

Chen,

Kálai,

Cascio

et al. 2023

Appl Magn Reson

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Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is an established tool for exploring protein structure and dynamics. Although nitroxide side chains attached to a single cysteine via a disulfide linkage are commonly employed in SDSL-EPR, their internal flexibility complicates applications to monitor slow internal motions in proteins and to structure determination by distance mapping. Moreover, the labile disulfide linkage prohibits the use of reducing agents often needed for protein stability. To enable the application of SDSL-EPR to the measurement of slow internal dynamics, new spin labels with hindered internal motion are desired. Here, we introduce a highly ordered nitroxide side chain, designated R9, attached at a single cysteine residue via a non-reducible thioether linkage. The reaction to introduce R9 is highly selective for solvent-exposed cysteine residues. Structures of R9 at two helical sites in T4 Lysozyme were determined by X-ray crystallography and the mobility in helical sequences was characterized by EPR spectral lineshape analysis, Saturation Transfer EPR, and Saturation Recovery EPR. In addition, interspin distance measurements between pairs of R9 residues are reported. Collectively, all data indicate that R9 will be useful for monitoring slow internal structural fluctuations, and applications to distance mapping via dipolar spectroscopy and relaxation enhancement methods are anticipated.

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Section: Discussionmentioning

confidence: 99%

A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins

Chen,

Kálai,

Cascio

et al. 2023

Appl Magn Reson

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Probing the Effects of Multisite Mutations in the Lipoic Acid Region of the BCOADC-E2 Protein

Wang,

Yang,

Wei

et al. 2024

IJMS

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Primary biliary cholangitis (PBC) is a chronic disease, the prevalence of which has been increasing in recent years. And the prevalence of patients who test negative with existing diagnostic techniques remains high. It was found that the antigenic BCOADC-E2 protein could detect patients with a negative original test. And experiments revealed that the lipoyl domain of BCOADC-E2 plays an important role. The present study was carried out to verify the necessity of maintaining the folding conformation of the lipoyl β-sheet of the protein in the lipoyl domain during the recognition of the BCOADC-E2 protein and the importance of the glutamic acid and isoleucine residues at position 4 and position 13, respectively. In order to search for a new pathway for the pre-detection of patients with PBC, firstly, the mutant proteins were subjected to an enzyme-linked immunosorbent assay (ELISA) with serum. Then, MTSSL spin tags were positioned at specific sites of the Cys mutant and reacted with serum samples from PBC patients and controls, and EPR spectroscopic data were measured. The multiple mutant proteins all reacted less specifically with the serum than the wild-type protein in the ELISA; the spectra measured for the pGEX-BCKD-E4A-I13A mutant were severely broadened, and the compactness at the conformational position of the lipoyl β-sheet structural conformation of the proteins of amino acids 4 and 13 remained unchanged. The EPR spectral data validate the importance of the glutamate and isoleucine residues at position 4 and position 13 and their necessity in the maintenance of the lipoyl β-sheet structural conformation of proteins in the lipoyl domain in anti-BCOADC-E2 recognition.

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Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label

Cited by 2 publications

References 47 publications

A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins

A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins

Probing the Effects of Multisite Mutations in the Lipoic Acid Region of the BCOADC-E2 Protein

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