An NMR strategy is described for measuring changes in C-13 spin-lattice relaxation times (T-1) of ligand molecules, at natural isotopic abundance, upon binding to macromolecules of potentially unlimited size. The rapidly reversible binding nature of a substrate-based inhibitor (BILN127SE, K-i = 5.4 muM) with the NS3 protease domain of the hepatitis C virus has been well documented and has served as an appropriate system for testing the transferred C-13 TI concept. C-13 T-1 relaxation, which is sensitive to motions that occur on the pico- to nanosecond time scale, were first measured for free BILN127SE. Upon addition of the protease at a 25:1 inhibitor-to-protease ratio, differential changes in the C-13 T-1 relaxation times of BILN127SE were observed. The equilibrium binding nature of the complex, results in a transfer of T-1 relaxation information of the ligand from the bound to the free state where it is more easily detected. The relative changes in C-13 T-1 relaxation provides a qualitative insight into the site-specific changes in ligand immobilization upon binding to the protease. Comparisons of this dynamics information are made with structural data deduced from H-1 NOESY, line-broadening, J-coupling, and ROESY experiments. The combination of dynamics and structural information should provide medicinal chemists with further opportunities to design more potent, chemically rigidified inhibitors.