Mn(ii) tags for DEER distance measurements in proteins via C–S attachment

Abstract

Mn(2+) chelating tags for Mn(2+)-Mn(2+) distance measurements by pulse EPR spectroscopy were developed. They feature a stable C-S conjugation to the protein, high reactivity towards cysteine thiols and short and rigid linkers that can be used in distance measurements with high resolution under reductive conditions. Double electron-electron resonance measurements at 95 GHz on ubiquitin labeled with these tags showed the expected narrow distance distribution.

A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

“…However, for MnDOTA, these effects were mostly obscured by the intrinsic flexibility of the macromolecules and/or the tether to the metal spin label . In contrast, the pseudosecular term contributions were expected to be much smaller for MnEDTA and MnPEDTA due to their larger and more distributed D values …”

“…As expected, in the corresponding Mn II −Mn II PELDOR measurements, λ values (≈0.7–1 %), which were comparable to or somewhat greater than that of MnEDTA, were observed. However, in contrast to MnEDTA, narrow distance distribution profiles, down to 0.6 nm full‐width at half‐height (fwhh), were observed in the MnPEDTA measurements; this was attributed to the short length and rigidity of the tether connecting the spin label to the protein . Previously, we reported the grafting of two [Mn(dota)] 2− (MnDOTA; DOTA 4− =1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetate; D =280(150) MHz) derivatives through a flexible succinimidyl thioether tether onto a series on polyproline spacers .…”

“…In the last decade, high‐spin gadolinium(III) (S=7/2) and manganese(II) (S=5/2) complexes have emerged as attractive alternatives . At higher fields, the main feature in their EPR spectra is the |−1/2>→|+1/2> “central transition”.…”

“…However, this is partially compensated for by the lower spin multiplicity of Mn II compared with that of Gd III . To date, only three Mn II −Mn II PELDOR studies have been reported and all were performed at the W‐band frequency . In the first instance, the distance between two [Mn(edta)] 2− (MnEDTA; EDTA 4− =ethylenediaminetetraacetate) derivatives, grafted through a disulfide tether onto cysteine residues of a protein fragment, was measured .…”

“…Recently, Martorana et al. reported the attachment of [Mn(pedta)] − derivatives (MnPEDTA; PEDTA 3− = N ‐(pyrid‐2‐ylmethyl)ethylenediamine‐ N , N′,N′ ‐triacetate) to cysteine residues of ubiquitin mutants through C−S conjugation . For two of the derivatives, the D values were 1860(900) and 3060(600) MHz; the values in parentheses denote their distributions.…”