NMR relaxation dispersion reveals an order of magnitude range of acid loop motions in protein tyrosine phosphatase 1B.

Motion of the active site acid loop is critical for the proper function of the enzyme, protein tyrosine phosphatase 1B (PTP1B). To investigate the motion of this loop, we incorporated 7-fluorotryptophan at residue 179 (7F-Trp) near the hinge of the acid loop - replacing the naturally occurring tryptophan at this position. Control experiments show identical kcat and Km values for for WT and 7F-Trp enzymes and 15N HSQC experiments show minimal chemical shift changes caused by the incorporation of fluorotryptophan into this enzyme. We then investigated the conformational motions of the acid loop by multifield 19F Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and R1ρ experiments. These experiments demonstrate that Trp179 has an exchange rate constant (kex) of 16,000 s-1 in the apo enzyme. This value contrasts with kex values for the amide backbone positions on the acid loop of 900 s-1 and sidechain methyl motions for V184 that occur with kex = 1800 s-1. These experiments demonstrate that the motion of the acid loop in PTP1B occurs on a rugged energy landscape governed by multiple processes. Furthermore, this work highlights the additional dynamical insight gained through the use of different spin-1/2 nuclei as motional probes.