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Abstract

Deuterated 3-trimethylsilylpropionic acid binding to bovine serum albumin was used as a model system to examine the feasibility and limitations of using the deuterium off-resonance rotating frame spin-lattice relaxation experiment for the study of equilibrium ligand-binding behavior to proteins. The results of this study demonstrate that the rotational-diffusion behavior of the bound species can be monitored directly, i.e., the observed correlation time of the ligand in the presence of a protein is approximately equal to the correlation time of the ligand in the bound state, provided that the fraction of bound ligand is at least 0.20. The presence of local ligand motion and/or chemical exchange contributions to relaxation in the bound state was inferred from the observation that the correlation time of the bound ligand was somewhat smaller than the correlation time characterizing the overall tumbling of the protein. An approximate value for the fraction of bound ligand was obtained from off-resonance relaxation experiments when supplemental spin-lattice or transverse relaxation times were employed in the analysis. Incorporation of local motion effects for the bound species into the theoretical relaxation formalism enabled the evaluation of an order parameter and an effective correlation time, which in conjunction with a wobbling in a cone model, provided additional information about ligand motion in the bound state.