Small neutral Gd(iii) tags for distance measurements in proteins by double electron–electron resonance experiments

Abstract

Spin labels containing a Gd(iii) ion have become important for measuring nanometer distances in proteins by double electron-electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(iii) tag used. Here we report the performance of two Gd(iii) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B-NS3 protease than previously established Gd(iii) tags. The improved performance is consistent with the absence of specific hydrophobic or charge-charge interactions with the protein. In the case of the Zika virus NS2B-NS3 protease, unexpectedly broad Gd(iii)-Gd(iii) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B-NS3 protease, both in the presence and absence of an inhibitor.

Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme

“…This is referred to as refolded AncCDT-1. The AzF residues were modified with propargyl-DO3A-Gd(III) tags as previously described (Figure S2a) 41,49 and mass spectrometry analysis indicated tag ligation yields of >80 % at each site.…”

“…This work expands the scope of the DEER approach 19,[72][73][74][75][76] for use in this context by using Gd(III) spin labels; it is the first time that the propargyl-DO3A-Gd(III)/DEER approach has been used to study rigid-body protein dynamics and it demonstrates the excellent performance of the propargyl-DO3A-Gd(III) tag. The tag is independent of cysteine residues as it can be ligated to AzF residues and the small size, lack of net charge and hydrophilic character of the DO3A-Gd(III) complex disfavours specific interactions with the protein surface, thus increasing the reliability of computational predictions of the Gd(III)-Gd(III) distance distributions obtained with this tag 41 . This study highlights its efficiency (the DEER measurements requiring only nanogram amounts of protein) in studying the protein dynamics of a series of states along an evolutionary trajectory and how it afforded distribution widths that were sufficiently narrow to observe the co-existence of open and closed protein conformations that differ by only about 1 nm, to show the presence of two conformations simultaneously sampled in solution and to detect ligand-induced conformational changes.…”

“…In this study, we investigated two plausible explanations for how remote mutations could have led to an increase in catalytic activity along this evolutionary trajectory: they may have (i) altered the sampling of rotamers of active site residues, such as the general acid Glu173, thereby changing the structure and character of the active site and controlling the configuration of active site residues, and/or (ii) altered the equilibrium between open and closed states of the protein to minimize sampling of the catalytically unproductive open state. To test these hypotheses, we used a combination of protein crystallography, MD simulations and DEER distance measurements on protein variants into which we incorporated the unnatural amino acid pazidophenylalanine (AzF) to allow biorthogonal conjugation, making it specific even in the presence of native cysteine residues, to a propargyl-DO3A-Gd(III) tag 41 . The Gd(III)-Gd(III) DEER measurements were carried out at W-band frequency (94.9…”

Optimising broadband pulses for DEER depends on concentration and distance range of interest

“…A crucial parameter for pulsed dipolar spectroscopy is the modulation-to-noise parameter MNR = λ n , with the modulation depth λ and the noise level n. We calculated the noise similarly to published procedures by the standard error from a fit with a smoothing spline (Bahrenberg et al, 2017;Breitgoff et al, 2019;Mentink-Vigier et al, 2013). We excluded the first 10 data points from the form factor because the spline typically showed some deviations at the start of the trace.…”

rDEER: A Modified DEER Sequence for Distance Measurements Using Shaped Pulses

“…As the majority of proteins and nucleic acids are diamagnetic, spin labels, which are covalently attached to the biomolecule of interest, are introduced via site-directed spin labeling (SDSL) [2][3][4]. While the most commonly used spin labels are nitroxide-based (mainly S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate, MTSSL), the use of other paramagnetic tags as spin labels for biomolecules like triarylmethyl [5,6], Cu(II) [7,8], Mn(II) [9][10][11][12][13] or Gd(III) [14][15][16][17][18][19][20][21][22] has been successfully demonstrated. In the case of these metal ions, the metal is usually coordinated to chelating tags that are covalently attached to the biomolecule.…”