Detection of Intermolecular NOEs in Small and Large Protein/Peptide Complexes

Weak protein-ligand interactions play important roles in biological recognition. Detection of pairwise interactions in such complexes is a major challenge for both X-ray crystallography and NMR. We developed two approaches that rely on transferred nuclear Overhauser effect, TRNOE, to detect such interactions.

TRNOE, in combination with asymmetric deuteration of a protein and a peptide ligand was used to detect intermolecular interactions in HIV-1 gp120 ternary complex with a CD4-mimic miniprotein and a 27-residue peptide corresponding to the N-terminal segment of the CCR5 chemokine receptor, Nt-CCR5(1-27). The TRNOE cross peaks in the ternary complex were assigned to the specific Tyr protons in Nt-CCR5(1-27). The TRNOE/asymmetric deuteration method benefits from the sensitivity of the NOESY experiment and does not suffer from the sensitivity losses associated with isotope edited/isotope filtering approaches that rely on magnetization transfer between protons and heteronuclei bonded to them. This technique can be widely applied for studying large protein complexes that exhibit fast off-rates.

In a following study, we demonstrated that TRNOE in combination with the ¹³C-edited/¹³C-filtered NOESY experiment can be used to study intermolecular interactions also in small and medium sized protein complexes. We used this approach to study the interactions of the chemokine RANTES with the Nt-CCR5(1-27) peptide. The TRNOE phenomenon led to more than doubling in the signal to noise ratios (SNR) for the intermolecular NOEs observed in the ¹³C-edited/¹³C-filtered experiment for the 11.5 kDa complex of monomeric RANTES with Nt-CCR5(1-27). An even better improvement in the SNR was achieved with dimeric RANTES complex with Nt-CCR5(1-27) (23 kDa). The isotope-edited/isotope filtered TRNOE spectrum has the potential to show all intermolecular interactions in the complex.