Anisotropic rotational diffusion in model-free analysis for a ternary DHFRcomplex

Model-free analysis has been extensively used to extract information on mot ions in proteins over a wide range of timescales from NMR relaxation data. We present a detailed analysis of the effects of rotational anisotropy on t he model-free analysis of a ternary complex for dihydrofolate reductase (DH FR). Our findings show that the small degree of anisotropy exhibited by DHF R (D-parallel to/ D-perpendicular to =1.18) introduces erroneous motional m odels, mostly exchange terms, to over 50% of the NH spins analyzed when iso tropic tumbling is assumed. Moreover, there is a systematic change in S-2, as large as 0.08 for some residues. The significant effects of anisotropic rotational diffusion on model-free motional parameters are in marked contra st to previous studies and are accentuated by lowering of the effective cor relation time using isotropic tumbling methods. This is caused by the prepo nderance of NH vectors aligned perpendicular to the principal diffusion ten sor axis and is readily detected because of the high quality of the relaxat ion data. A novel procedure, COPED (COmparison of Predicted and Experimenta l Diffusion tensors) is presented for distinguishing genuine motions from t he effects of anisotropy by comparing experimental relaxation data and data predicted from hydrodynamic analyses. The procedure shows excellent agreem ent with the slow motions detected from the axially symmetric model-free an alysis and represents an independent procedure for determining rotational d iffusion and slow motions that can confirm or refute established procedures that rely on relaxation data. Our findings show that neglect of even small degrees of rotational diffusion anisotropy can introduce significant error s in model-free analysis when the data is of high quality. These errors can hinder our understanding of the role of internal motions in protein functi on.