¹⁹F Paramagnetic Relaxation Enhancement: A Valuable Tool for Distance Measurements in Proteins

Abstract

Fluorine NMR paramagnetic relaxation enhancement was evaluated as a versatile approach for extracting distance information in selectively F-labeled proteins. Proof of concept and initial applications are presented for the HIV-inactivating lectin Cyanovirin-N. Single F atoms were introduced at the 4-, 5-, 6-or 7 positions of Trp49 and the 4 position of Phe4, Phe54 and Phe80. The paramagnetic MTSL label was attached to Cys residues that were placed into the protein at positions 50 or 52. 19 F-T 2 NMR spectra with different relaxation delays were recorded and the transverse 19 F-PRE rate, 19 F-Γ 2 , was used to determine the average distance between the F nucleus and the paramagnetic center. Our data show that experimental 19 F PRE based distances correspond to ~0.93 of the 1 H N -PRE distances, in perfect agreement with the gyromagnetic γ 19 F/γ 1 H ratio, thereby demonstrating that 19 F PREs are excellent alternative parameters for quantitative distance measurements in selectively F-labeled proteins. Keywords fluorine; paramagnetic; NMR; distance; protein In Paramagnetic Relaxation Enhancement (PRE) NMR experiments the increase in longitudinal or transverse relaxation rates, induced by the presence of a paramagnetic center, is measured. The effect is <r −6 > distance dependent, and, as a result, information on the distance between the paramagnetic moiety to the measured nuclei is obtained.In biomolecular NMR, PREs are increasingly used for providing long-range distance information that complements nuclear Overhauser effect (NOE)-derived, short (≤ 5 Å) interproton distance restraints. In cases where NOE data were found limited, distances derived from PREs provided valuable supplemental restraints and permitted either characterization of the global folds for some proteins, [1,2] or delineate structural properties of disorder proteins. [3,4] Correspondence to: Angela M. Gronenborn, amg100@pitt.edu. Supporting information for this article is given via a link at the end of the document. HHS Public AccessAuthor manuscript Angew Chem Int Ed Engl. Author manuscript; available in PMC 2017 January 04. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe most widely used spin label, originally developed for EPR studies, [5,6] is the nitroxide radical MTSL (1-oxyl-2,2,5,5-tetramethyl-Δ 3 -pyrroline-3-methyl)methanethiosulfonate. [7,8] It enhances the transverse relaxation rate (R 2 ) of protons and is introduced into proteins via site specific labeling of cysteine residues. [9][10][11] PRE measurements in proteins commonly monitor 1 H line broadening or intensity attenuation of proton resonances, caused by R 2 relaxation rate enhancements, and these are used to derive distances, with an accessible distance range for the MTSL spin label to protons of 13-25 Å. Resonances of residue in close proximity to the spin label are broadened beyond detection, while for long distances, the effect becomes undetectable.

A Metastable Contact and Structural Disorder in the Estrogen Receptor Transactivation Domain

“…Through the multi-technique data integration and contact map analyses, we identified long-range interactions involving residues S118 and I33 driving compactness within the ensemble and showed that S118 mutation mediates conformational changes and alters coactivator binding. Furthermore, through the means of fluorine-19 ( 19 F) NMR (Chrisman et al, 2018;Didenko et al, 2013;Kitevski-LeBlanc and Prosser, 2012;Li et al, 2010;Matei and Gronenborn, 2016), we show that mutations near residue I33 alter 19 F chemical shifts at the position of residue S118, thereby confirming the proposed metastable I33-S118 contact in the ensemble. Overall, our multi-technique biophysical approach allowed us to elucidate specific structural features of the ERa-NTD that mediate its transactivation function, and our findings raise important questions as to the potential of ''contact metastability'' to mediate functions for other nuclear receptors and/or IDPs in general.…”

“…To test the validity of the proposed I33-S118 interaction, we conducted 19 F NMR studies on the NTD by introducing a cysteine residue at the location of S118 to allow covalently linked 19 F labeling with a trifluoromethyl (-CF 3 ) group (depicted in Figures 7A and 7B). The 19 F labeling was achieved using 3-bromo-1,1,1-trifluoroacetone (BTFA), a widely used labeling reagent in 19 F NMR studies of both disordered and well-folded proteins (Didenko et al, 2013;Kitevski-LeBlanc and Prosser, 2012;Li et al, 2010;Matei and Gronenborn, 2016), with a more recent application to the nuclear receptor PPARg (Chrisman et al, 2018). Hereafter, we refer to the BTFA-labeled NTD S118C protein variant as NTD S118C -BTFA.…”

Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog

“…Its magnitude is described by the Solomon−Bloembergen equations 17 , 18 : where γ is the nuclear gyromagnetic ratio and J MF is the spectral density function accounting for the local side chain motions ( Supplementary Note ). Because PRE depends quadratically on the gyromagnetic ratio, it is weak for 13 C and 15 N nuclei but is significant for 1 H and 19 F 19 . Retrieving the distance-dependent paramagnetic component from the R 1 PRE for 1 H nuclei is difficult because of the interference from 1 H- 1 H cross relaxation 15 .…”

A suite of 19F based relaxation dispersion experiments to assess biomolecular motions

“…Recently, fluorine ( 19 F) NMR has regained attraction (Hellmich et al 2009 ; Liu et al 2012 ; Kim et al 2013 ; Kitevski-Leblanc et al 2013 ; Aramini et al 2014 ; Hoang and Prosser 2014 ; Manglik et al 2015 ; Matei and Gronenborn 2016 ; Lu et al 2019 ; Huang et al 2020 ). This spin is absent from virtually all biomolecules, however, 19 F probes can be artificially introduced into proteins by incorporation of fluorinated amino acids (Crowley et al 2012 ) or through post-translational modification with fluorine-containing tags (Brauer and Sykes 1986 ).…”