Theoretical Basis for 1D Proton Line Broadening NMR and 1H-15N HSQC NMR.

1D proton line broadening studies were performed to monitor binding of PAR3/PAR3G (44–56) peptide fragment to prothrombin versus thrombin.^{43, 45, 46} Protein-peptide complexes were prepared with a 10-fold excess of peptide. Peptide protons that undergo interactions experience transient on/off events that cause an increase in the proton transverse relaxation rate and/or cause changes in the observed chemical shift position depending on the timescale of the interaction. The resultant alterations in peptide proton line width/shape reflect the weighted contributions of bound and free populations. This NMR line broadening approach can help map the peptide residue protons that come in direct contact with the protein surface.

¹H-¹⁵N HSQC NMR titrations were used to assess whether specific ¹⁵N-labeled residues located within a peptide ligand exhibited fast, intermediate, or slow exchange on/off a protein surface.⁴⁷ In these titrations, changes in chemical shift are monitored as a function of protein-ligand ratios. For weak binding, the interaction is under the fast exchange regime (k_ex >> |Δω|) where k_ex is the exchange rate of the interaction and Δω is the resonance frequency difference between the bound and free states. Only one signal per ¹⁵N-labeled residue is observed reflecting the population-weighted average of bound and free states in terms of chemical shift, intensity, and linewidth. Furthermore, NMR chemical shifts can be used to quantitatively evaluate affinities for individual ¹⁵N labeled residues.^{48, 49} For the moderate affinity, intermediate exchange regime (k_ex ≈ |Δω|), the ¹⁵N peak may disappear during the titration due to extensive line broadening and upon increasing the bound population will later reappear. Finally, for tight binding, in which the interaction is described by the slow exchange regime (k_ex << |Δω|), two sets of resonances are observed: one corresponding to the free and the other to the bound state. Under this situation, the change in the intensity of the peaks will correspond to the relative populations of each state during the course of the titration.