Global Dynamics and Exchange Kinetics of a Protein on the Surface of Nanoparticles Revealed by Relaxation-Based Solution NMR Spectroscopy

Abstract

The global motions and exchange kinetics of a model protein, ubiquitin, bound to the surface of negatively charged lipid-based nanoparticles (liposomes) are derived from combined analysis of exchange lifetime broadening arising from binding to nanoparticles of differing size. The relative contributions of residence time and rotational tumbling to the total effective correlation time of the bound protein are modulated by nanoparticle size, thereby permitting the various motional and exchange parameters to be determined. The residence time of ubiquitin bound to the surface of both large and small unilamellar liposomes is ∼20 μs. Bound ubiquitin undergoes internal rotation about an axis approximately perpendicular to the lipid surface on a low microsecond time scale (∼2 μs), while simultaneously wobbling in a cone of semiangle 30-55° centered about the internal rotation axis on the nanosecond time scale. The binding interface of ubiquitin with liposomes is mapped by intermolecular paramagnetic relaxation enhancement using Gd(3+)-tagged vesicles, to a predominantly positively charged surface orthogonal to the internal rotation axis.

Figure 1

¹⁵N–ΔR₂ profiles for 0.8 mM U-[¹⁵N/²H] ubiquitin (dissolved in H₂O) in the presence of a 1:2 ratio (mol/mol on a lipid molecule basis) negatively charged (POPG, filled symbols) and zwitterionic (POPC, open symbols) liposomes of differing size: (A) LUVs (blue) and (B) SUVs (green). ¹⁵N–ΔR₂ dependence on magnetic field strength for (C) LUVs and (D) SUVs, and (E) on nanoparticle size. The black lines in C–E represent simulated correlations derived from global best-fitting of all LUV and SUV ¹⁵N–ΔR₂ data (see text, Figure 2 and SI). Experimental temperature = 25 °C.

Figure 2

Global dynamics of ubiquitin on the surface of liposome nanoparticles. (A) Ubiquitin (gray ribbon, PDB 1UBQ) rotates about an internal rotation axis (denoted as r and displayed in red), shown as perpendicular to the surface of the lipid bilayer (blue) while wobbling in a cone of semiangle β₀. (B) Relationship between the axis r (red) and the x, y, and z axes of the inertia tensor (blue, used as an arbitrary internal reference frame): θ is the angle between the r and z axes; and φ the angle (not shown) between the x axis and the projection of the r axis on the x–y plane. Dependence of ¹⁵N–ΔR₂ in the presence of (C) LUVs and (D) SUVs on the angle α between the N–H bond vectors and the internal rotation axis: experimental data are shown as blue circles; the best-fit curves, from global fitting to all LUV and SUV data, are in red (S_w² = 0.5 and τ_w = 300 ns; see text and SI).

Figure 3

Proton intermolecular transverse PREs (Γ₂) observed for U-[¹⁵N/²H]/[¹H_methyl/¹³C_methyl-ILV] ubiquitin in the presence of negatively charged Gd³⁺-tagged POPG LUVs. (A) ¹H-Γ₂ profiles for backbone amide (top) and Ile/Leu/Val methyl (bottom) protons. (B) PRE mapping of the interaction surface of ubiquitin. The internal rotation axis (red dot) is perpendicular to the plane of the figure and orthogonal to the view shown in Figure 2A. ¹H_N-Γ₂ PREs are color-coded from purple (110 s⁻¹) to white (background = 40 s⁻¹), and residues with ¹H_N-Γ₂ ≥ 77 s⁻¹ are labeled; residues with large (>80 s⁻¹) ¹H_methyl-Γ₂ values are colored in green. (C) Molecular surface of ubiquitin (same views as in panel B) color-coded according to electrostatic potential (±5 kT with blue, positive; white, neutral; and red, negative). Lipid molecules on the nanoparticle surface are shown schematically as gray spheres.