Nanometer-Scale Distance Measurements in Proteins Using Gd³⁺ Spin Labeling

Abstract

Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function. In this work we introduce Gd3+ spin labeling for nanometer-range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPR). To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and τC14, labeled at strategically selected sites with either two nitroxides or two Gd3+ spin labels. In analogy to conventional site-directed spin labeling using nitroxides, Gd3+ tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups. Measurements were carried out on X-band (∼9.5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd3+-labeled proteins. In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd3+−Gd3+ distance distribution had a maximum at 2.9 nm, as compared to 2.5 nm for the nitroxides. In the protein τC14, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide−nitroxide distance distribution with a maximum at 2.5 nm, and the W-band measurements gave a Gd3+−Gd3+ distance distribution with a maximum at 3.4 nm. The Gd3+−Gd3+ distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd3+ labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels. Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection.

Gd(III)–Gd(III) distance measurements with chirp pump pulses

“…The stock solutions of Gd-ruler 1 9 and 1 11 had concentrations of 2 mM in D 2 O (pH 8.0), containing 20 mM NaCl. For the EPR experiments, the stock solutions were diluted with D 2 O and glycerol-d 8 (1:1 in volume). For Gd-ruler 1 3 , 20 µl of a solution with a concentration of 600 µM was used.…”

“…Modifications of this approach involve the use of non-identical spin labels, in particular a Gd(III) center and a nitroxide [3,4], as well as different experimental schemes, such as relaxation-induced dipolar modulation (RIDME) [5] or continuous-wave EPR [6]. Distance measurements involving Gd spin labels have been successfully demonstrated on a number of systems, including model compounds, peptides, nanoparticles, proteins and DNAs [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Moreover, a promising biochemical property of Gd spin labels is the stability under reducing conditions, which recently enabled Gd-Gd distance measurements of peptides and proteins embedded in cellular environments [22,23].…”

Nanometer-Range Distance Measurement in a Protein Using Mn2+ Tags

“…The corresponding nitroxide−nitroxide distance was much shorter (2.5 nm), which must be attributed to nonuniform sampling of the conformational space of the tag, possibly arising from preferential hydrophobic interactions with the protein. 13 For comparable D values, DEER measurements are expected to be more sensitive for Gd 3+ than Mn 2+ tags because the central line of the Mn 2+ spectrum is split into six 55 Mn hyperfine lines, thus reducing both λ and V 0 . This effect, however, is partly compensated for by the about 2-fold greater population difference of the central transition for Mn 2+ in the temperature range of 6−10 K ( Figure S2, Supporting Information).…”

“…The resulting orientation selection complicates data analysis when the two nitroxides are motionally constrained, requiring the determination of five Euler angles in addition to the electron− electron distance to fit the data. 13,20,21 Here we introduce a new family of spin labels based on Mn 2+ chelates for long-range distance measurements in proteins at high magnetic fields. Mn 2+ shares many of the spectroscopic advantages of Gd 3+ and, by virtue of its different coordination chemistry and lesser toxicity in living systems, significantly broadens the scope of suitable metal tags.…”

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

“…Coupled with site-directed spin-labeling (SDSL), EPR is oftentimes used to characterize protein and nucleic acid structures and dynamics, conformational changes, molecule folding, macromolecule complexes, and oligomeric structures [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]17]. The majority of biomolecules do not contain unpaired electrons from which one can obtain an EPR signal; therefore, spin-labeling approaches have been developed [15,[18][19][20][21][22][23][24][25] where site-specific persistent radicals or paramagnetic metal-probes are incorporated at specific locations within a biomolecule. Properties of the EPR spectra that originate from these probes, positioned at welldefined vantage points, provide structural and dynamic constraints.…”