Theory and experimental results of transfer NOE experiments. II. The influence of residual mobility and relaxation centers inside the protein on the size of transfer NOEs

Abstract

Experimental evidence is presented for a strong dependence of transfer NOE values on the Larmor frequency for certain residues of a peptide in its complex with a protein. This dependency has been explained by residual mobility in these residues of the peptide when bound. As a consequence, effective correlation times exist for some proton-proton pairs that are at least one order of magnitude smaller than expected for a rigid complex. This is shown to be the main reason for the failure to observe certain transfer NOEs in such a complex when studied at proton frequencies of 400 MHz or less. Another factor that can reduce the size of transfer NOES is the possibility of leakage of magnetization toward relaxation centers inside the protein. We have observed several intermolecular NOE cross peaks demonstrating such an intermolecular exchange of magnetization. The influence of cross relaxation to relaxation centers in the protein on the buildup and magnitude of proton transfer NOES for the bound and the free ligand is described in a model of the ligand-protein system that consists of a set of Bloch equations in which chemical exchange, transfer of magnetization, and relaxation sinks in the protein have been included. Simulations have been done for the cases of fast and intermediate exchange on the spin-lattice relaxation time scale while in all cases bound and free ligand signals were assumed to be in fast exchange on the chemical-shift scale. It is found that the magnitude of intramolecular transfer NOEs is reduced by the presence of cross relaxation from the peptide into the protein, in particular when the protein has relaxation centers such as methyl groups and aromatic rings. It is shown that these effects can reduce the size of transfer NOE effects by an order of magnitude for those ligand protons that are directly in contact with the protein. The simulations for ligand protein concentration ratios of 2, 10, and 100 with various numbers of relaxation sinks in the protein show that concentration ratios and mixing times must be optimized in order to observe sizeable transfer NOES in 2D NMR experiments. The influence of these effects on the accuracy of distances determined via transfer NOEs is also discussed.