Identification and removal of nitroxide spin label contaminant: impact on PRE studies of α-helical membrane proteins in detergent

Abstract

NMR paramagnetic relaxation enhancement (PRE) provides long-range distance constraints (~15-25 Å) that can be critical to determining overall protein topology, especially where long-range NOE information is unavailable such as in the case of larger proteins that require deuteration. However, several challenges currently limit the use of NMR PRE for α-helical membrane proteins. One challenge is the nonspecific association of the nitroxide spin label to the protein-detergent complex that can result in spurious PRE derived distance restraints. The effect of the nitroxide spin label contaminant is evaluated and quantified and a robust method for the removal of the contaminant is provided to advance the application of PRE restraints to membrane protein NMR structure determination.

Figure 1

EPR spectra of wt TM0026 (A) and V49R1 (B) at various stages of spin label removal. The use of a desalting PD-10 column (left panel) effectively removes the bulk of unreacted spin label; however, it is ineffective at removing unreacted spin label associated with the protein micelle complex. After 3 days of incubation at room temperature and passing through a Co2+ IMAC column (right panel), the contaminate spin label concentration is reduced eight fold. C and D: 15N, 1H-TROSY-HSQC spectra of wt TM0026 after attempts to remove excess MTSSL and corresponding plots of intensity ratios versus residue are shown: (C) after a PD-10 desalting column was used to remove excess spin label (MTSSL) and (D) after 3 days at room temperature and a Co2+ IMAC column to remove excess spin label (intensities plotted below are normalized to TM0026 without spin label added). A black line is drawn at an intensity ratio of 0.85, a commonly used upper limit for quantitative distance restraints.

Figure 2

Labeling efficiency quantified using MALDI TOF mass spectrometry for four sites on TM0026. All four sites are on transmembrane α-helices as assessed by the predicted topology and NOE crosspeaks between the amide proton and the alkyl chain of the detergent (Supporting Information Fig. S2). In addition, the EPR spectra of V15R1 and A13R1 indicate the nitroxide has restricted motion, which suggests these residues are tertiary contact sites and more occluded compared to L14R1 and L51R1. (The labeling efficiency is <100%; however, disulfide bonds fragmentation during the MALDI TOF measurement was previously observed and could account for the ∼10% of under-labeling observed).

Figure 3

The 15N, 1H-TROSY-HSQC spectra of wt TM0026 with varying levels of MTSSL titrated into solution and corresponding intensity ratios versus residue plotted below: (A) 1:3 molar ratio of wt TM0026:MTSSL; (B) 1:6 molar ratio of wt TM0026:MTSSL; (C) 1:15 molar ratio of wt TM0026:MTSSL. The two tryptophan indole peaks are shown as an inset. A black line is drawn at an intensity ratio of 0.85, a commonly used upper limit for quantitative distance restraints.