Mapping Membrane Protein Backbone Dynamics: A Comparison of Site-Directed Spin Labeling with NMR 15N-Relaxation Measurements

Abstract

The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR (15)N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.

Paramagnetic Chemical Probes for Studying Biological Macromolecules

“…protein domain studies 695,696 protein alignment 697 protein−ligand interaction 698 RNA structure analysis 699 protein structure refinement 701 M8-CAM-I (Figure 9, 12) protein accessible surface characterizations 345,346,355,[711][712][713][714]732,733 commercially available and frequently used but somewhat hydrophobic; unstable under reducing conditions BPTI aggregation 715 protein folding 347 protein dynamics studies [664][665][666][667]670,730 protein distance measurements by 19 F-PRE 587 Gd(III)DTPA-BMA (Figure 13) protein surface accessible regions characterizations 350,716,734 high solubility, stability, and low protein binding affinity; zero net charge BPTI aggregation 715 protein structure determination 352,721 protein complexes studies 719,723 protein domain studies 722 spectral editing 726 Gd(III)TTHA-TMA (Figure 13) protein dynamic determinations 353 zero net charge [Ln(III)(DOTP)] 5− (Figure 13) protein−protein interaction 718 high negative charge; can generate both PCSs (loaded with Tb(III)) and PREs (loaded with Gd(III)) in some cases [Ln(III)(DPA) 3 ] 3− (Figure 13) multisite protein−ligand binding interactions 724 can generate both PCSs (loaded with Tb(III)) and PREs (loaded with Gd(III)) in some cases cationic peptides structure determination 725…”

“…667 MTSL has also been utilized in NMR studies of proteins in the solid state, 649,652,668,669 where structural restraints from PREs can amount to a sizable fraction of the data available for structure calculations. Especially in the case of membrane proteins, MTSL has proven useful to assess their dynamics, 670 structure, 586,671 and function. 672 Using the MTSL tagged and cyanobacterial lectin CV−Ncontaining fluorinated amino acids as an example, Matei and Gronenborn demonstrated that the high resolution of 19 F NMR spectra allows facile measurement of the distances between 19 F spins and the paramagnetic center by 19 F PREs, which proved to be in a good agreement with the results from 1 H PRE measurements.…”

“…MTSL has also been utilized in NMR studies of proteins in the solid state, ,,, where structural restraints from PREs can amount to a sizable fraction of the data available for structure calculations. Especially in the case of membrane proteins, MTSL has proven useful to assess their dynamics, structure, , and function …”

Structural and dynamic origins of ESR lineshapes in spin-labeled GB1 domain: the insights from spin dynamics simulations based on long MD trajectories

“…Furthermore, backbone motions are not directly transmitted to the proxyl ring because their effect is damped by the intervening flexible R1 side chain. While SDSL ESR can successfully discriminate between protein order and disorder [101][102][103][104] , it is not well suited to quantify small variations in S 2 within the fairly rigid protein scaffold.…”