Heaven, G, Hollas, MA, Tabernero, L and Fielding, AJ (2021) Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label. Applied Magnetic Resonance. ISSN 0937-9347

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Abstract

Structural investigations of proteins and their biological complexes are now frequently complemented by distance constraints between spin labeled cysteines generated using double electron–electron resonance (DEER) spectroscopy, via site directed spin labeling (SDSL). Methanethiosulfonate spin label (MTSSL), has become ubiquitous in the SDSL of proteins, however, has limitations owing to its high number of rotamers, and reducibility. In this article we introduce the use of bromoacrylaldehyde spin label (BASL) as a cysteine spin label, demonstrating an advantage over MTSSL due to its increased selectivity for surface cysteines, eliminating the need to ‘knock out’ superfluous cysteine residues. Applied to the multidomain protein, His domain protein tyrosine phosphatase (HD-PTP), we show that BASL can be easily added in excess with selective labeling, whereas MTSSL causes protein precipitation. Furthermore, using DEER, we were able to measure a single cysteine pair distance in a three cysteine domain within HD-PTP. The label has a further advantage of comprising a sulfide in a three-bond tether, making it a candidate for protein binding and in-cell studies.

Item Type:	Article
Uncontrolled Keywords:	0202 Atomic, Molecular, Nuclear, Particle and Plasma Physics, 0301 Analytical Chemistry, 0306 Physical Chemistry (incl. Structural)
Subjects:	Q Science > QC Physics Q Science > QD Chemistry
Divisions:	Pharmacy & Biomolecular Sciences
Publisher:	Springer
Date Deposited:	06 Jul 2021 10:40
Last Modified:	04 Sep 2021 05:16
DOI or ID number:	10.1007/s00723-021-01350-1
URI:	https://researchonline.ljmu.ac.uk/id/eprint/15237

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