F-19 Paramagnetic Relaxation Enhancement: AValuable Tool for Distance Measurements in Proteins

摘要

Fluorine NMR paramagnetic relaxation enhancement was evaluated as a versatile approach for extracting distance information in selectively F-labeled proteins. Proof of concept and initial applications are presented for the HIVinactivating lectin cyanovirin-N. Single F atoms were introduced at the 4-, 5-, 6-or 7 positions of Trp49 and the 4-position of Phe4, Phe54, and Phe80. The paramagnetic nitroxide spin label was attached to Cys residues that were placed into the protein at positions 50 or 52. F-19-T-2 NMR spectra with different relaxation delays were recorded and the transverse F-19-PRE rate, F-19-G2, was used to determine the average distance between the F nucleus and the paramagnetic center. Our data show that experimental F-19 PRE-based distances correspond to 0.93 of the H-1(N)-PRE distances, in perfect agreement with the gyromagnetic gamma F-19/gamma H-1 ratio, thereby demonstrating that F-19 PREs are excellent alternative parameters for quantitative distance measurements in selectively F-labeled proteins.