A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data

Fawzi, Nicolas L. and Fleissner, Mark R. and Anthis, Nicholas J. and Kálai, Tamás and Hideg, Kálmán and Hubbell, Wayne L. and Clore, Marius (2011) A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data. Journal of Biomolecular NMR, 51. pp. 105-114. ISSN 0925-2738


Download (503kB) | Preview


The measurement of 1H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metalchelating paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel, disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed α-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed α-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label.

Item Type: Article
Subjects: Q Science / természettudomány > QC Physics / fizika > QC04 Electricity. Magnetism. Electromagnetism / villamosság, mágnesesség, elektromágnessesség
Q Science / természettudomány > QD Chemistry / kémia > QD02 Physical chemistry / fizikai kémia
Depositing User: Dr. Tamás Kálai
Date Deposited: 15 Jan 2015 14:15
Last Modified: 03 Apr 2023 08:23

Actions (login required)

Edit Item Edit Item