Towards interpretation of intermolecular paramagnetic relaxation enhancement outside the fast exchange limit

Abstract

In an exchanging system between major and minor species, the transverse paramagnetic relaxation enhancement rate observed on the resonances of the major species (Γ 2 (app) ) is dependent upon the exchange regime between the species. Quantitative analysis of PRE data in such systems typically assumes that the overall exchange rate k ex between the species is fast on the PRE time scale (k ex ≫ Γ2). Recently, we have characterized the kinetics of binding of the model protein ubiquitin to large (LUV) and small (SUV) unilamellar lipid-based nanoparticles or liposomes (Ceccon A, Tugarinov V, Bax A, Clore GM (2016). J Am Chem Soc 138:5789-5792). Building upon these results and taking advantage of a strong paramagnetic agent with an isotropic g-tensor, Gd(3+), we were able to measure intermolecular methyl carbon and proton PREs between paramagnetically-tagged liposomes and ubiquitin. In the limit of fast exchange (k ex ≫ Γ2) the ratio of the apparent proton to carbon methyl PREs, ((1)Hm-Γ 2 (app) )/((13)Cm-Γ 2 (app) ), is equal to the square of the ratio of the gyromagnetic ratios of the two nuclei, (γΗ/γC)(2). However, outside the fast exchange regime, under intermediate exchange conditions (e.g. when Γ2 is comparable in magnitude to k ex) the ((1)Hm-Γ 2 (app) )/((13)Cm-Γ 2 (app) ) ratio provides a reliable measure of the 'true' methyl PREs.

Specific Interaction Sites Determine Differential Adsorption of Protein Structural Isomers on Nanoparticle Surfaces

“…Recent studies demonstrated the possibility of obtaining high‐resolution detail of nano‐bio interfaces . It emerged that simple protein molecules could be adsorbed on NPs at specific binding sites, in analogy with the mode of recognition of natural protein partners.…”

Probing the Binding Modes of a Multidomain Protein to Lipid-based Nanoparticles by Relaxation-based NMR

“…Therefore, to preserve the robustness of analysis for all domains and linkage types of Ub 2 and to enable direct comparison of the dynamics parameters obtained for different Ub 2 domains, we chose to determine the values of k on app,LUV for the different Ub 2 –LUV interactions independently from paramagnetic relaxation enhancement (PRE) experiments (Figure ). Outside the fast exchange regime ( k ex < Γ 2 , where Γ 2 is the PRE rate in the bound species), the maximal observed PRE, 1 H N –Γ 2,max app , after background subtraction, approaches a limiting value that is a factor of ∼1.3 lower than k on app,LUV for the exchange parameters previously established for Ub 1 . The values of k on app,LUV thus estimated from 1 H N –Γ 2,max app for each Ub 2 domain are reported in the first row of Table , which summarizes the parameters of exchange and dynamics obtained by combined analysis of LUV and SUV Δ R 2 data for the distal/proximal domains of K63- and K48-linked Ub 2 .…”

“…Therefore, to preserve the robustness of analysis for all domains and linkage types of Ub 2 and to enable direct comparison of the dynamics parameters obtained for different Ub 2 domains, we chose to determine the values of k on app,LUV for the different Ub 2 − LUV interactions independently from paramagnetic relaxation enhancement (PRE) experiments (Figure 2). 16 Outside the fast exchange regime (k ex < Γ 2 , where Γ 2 is the PRE rate in the The PRE background, due to diffusion of Ub 2 in the magnetic field generated by Gd 3+ -tagged LUVs, 3 is shown as a dashed line. The most significant PREs above background are shaded in gray.…”

“…bound species), the maximal observed PRE, 1 H N −Γ 2,max app , after background subtraction, 3 a limiting value that is a factor of ∼1.3 lower than k on app,LUV for the exchange parameters previously established for Ub 1 . 16 The values of k on app,LUV thus estimated from 1 H N −Γ 2,max app for each Ub 2 domain are reported in the first row of Table 1, which summarizes the parameters of exchange and dynamics obtained by combined analysis of LUV and SUV ΔR 2 data for the distal/proximal domains of K63-and K48-linked Ub 2 .…”