Research ArticleBIOPHYSICS

Functional protein dynamics on uncharted time scales detected by nanoparticle-assisted NMR spin relaxation

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Science Advances  14 Aug 2019:
Vol. 5, no. 8, eaax5560
DOI: 10.1126/sciadv.aax5560
  • Fig. 1 Protein dynamics into the hundreds of nanosecond to microsecond range accessible to NP-assisted solution NMR.

    (A) Protein molecules are in fast exchange between their rapidly tumbling free state and a slowly tumbling NP-bound state giving rise to effective transverse spin relaxation rates R2NP versus R2freein the absence of NPs. (B) Simulated dependence of R2free in the absence of NPs (blue) and ΔR2=R2NPR2free in the presence and absence of NPs (red) on the internal correlation time τi and motional restriction (S2 order parameter), which demonstrates the wide range of time scales sensitively probed by ΔR2. The blue and red curves were normalized by setting their maximal values to 1.0.

  • Fig. 2 Dynamics of Im7 protein by backbone 15N-NMR spin relaxation and molecular dynamics (MD) simulations.

    (A) 15N-R2 relaxation rates measured in the absence (gray) and presence (black) of NPs. Data points with asterisks (*) indicate substantial chemical exchange Rex effects. (B) R2 differences (ΔR2) of (A) with secondary structure of Im7 indicated at the bottom (4 α helices and 310 helix at N terminus). Experimental uncertainty (1 SD) is depicted by the shaded red area based on five independently measured ΔR2 profiles (see fig. S6). (C) Comparison of ΔR2-derived S2 (red circles) with standard model-free S2 order parameters (blue circles) and S2 values determined from 1-μs MD trajectory with variable averaging time window (from 250 ps to 1 μs). (D) S2R2) values mapped on three-dimensional (3D) crystal structure [Protein Data Bank (PDB) code 1AYI] show loops and tails that undergo substantial dynamics on pico- to microsecond time scales. N.A., not available.

  • Fig. 3 Dynamics of CBD1 protein domain from backbone 15N-NMR spin relaxation and MD simulations.

    (A) 15N-R2 relaxation rates measured in the absence (gray) and presence (black) of NPs. Gly399 (*) shows substantial chemical exchange Rex. (B) R2 differences (ΔR2) of (A) with secondary structure of CBD1 indicated at the bottom (nine β strands). (C) Comparison of ΔR2-derived S2 (red circles) with standard model-free S2 order parameters (blue circles) and S2 values determined from 1-μs MD trajectory with variable averaging time window (from 250 ps to 1 μs). (D) S2R2) values mapped on the 3D crystal structure of CBD1 (PDB code 2DPK) show loops and tails that undergo substantial dynamics on pico- to microsecond time scales.

  • Fig. 4 Visualization of complex loop motions by 1-μs MD trajectories.

    (A and C) Backbone dihedral angle–based PCA score plots of (A) Loop I of Im7 and (C) Loop E-F of CBD1 display multiple distinct conformational loop clusters. (B and D) 3D visualization of three cluster centers for each protein indicated by colored diamond (♦) symbols in score plots.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaax5560/DC1

    Supplementary Materials and Methods

    Fig. S1. Simulated dependence of R2free and ΔR2 on internal correlation time τi and S2 order parameter.

    Fig. S2. Range of validity of Eq. 3 for the extraction of S2 from ΔR2.

    Fig. S3. Experimental 15N spin relaxation parameters of Im7 in the absence of NPs.

    Fig. S4. Experimental 15N spin relaxation parameters of CBD1 in the absence of NPs.

    Fig. S5. Comparison between NMR S2R2) and x-ray B-factors of backbone nitrogen atoms in crystal structures.

    Fig. S6. Dependence of ΔR2 values on SNP concentration.

    Fig. S7. Mapping of experimental S2R2) onto the structural model of Im7 when bound to the DNase domain of colicin E7.

    References (4963)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Simulated dependence of R2free and ΔR2 on internal correlation time τi and S2 order parameter.
    • Fig. S2. Range of validity of Eq. 3 for the extraction of S2 from ΔR2.
    • Fig. S3. Experimental 15N spin relaxation parameters of Im7 in the absence of NPs.
    • Fig. S4. Experimental 15N spin relaxation parameters of CBD1 in the absence of NPs.
    • Fig. S5. Comparison between NMR S2R2) and x-ray B-factors of backbone nitrogen atoms in crystal structures.
    • Fig. S6. Dependence of ΔR2 values on SNP concentration.
    • Fig. S7. Mapping of experimental S2R2) onto the structural model of Im7 when bound to the DNase domain of colicin E7.
    • References (4963)

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