A quantitative, diffusion NMR based analytical tool to distinguish folded, disordered and denatured biomolecules.

The key questions in folding studies are the protein dimensions and the degree of folding. These properties are best characterized by the self diffusion coefficients D determining the hydrodynamic dimensions. In our present study, we derive empirical variations of D as a function of molecular mass M that distinguish folded, intrinsically disordered, and urea-denatured biomolecules. Reliable D values are obtained from diffusion NMR measurements performed under identical conditions using a representative set of proteins/peptides with diverse amino acid sequence and length. The established relations are easy to use analytical tools for molecular mass analysis and aggregation studies as well. Deriving equations under denaturing conditions has several pitfalls, and here, we provide a simple quantitative method for estimating the debated end point of denaturation, while already the 1D H-1 spectrum gives a qualitative picture of the collapsed, denatured structure. Data indicate that the intrinsically disordered proteins have a similar behavior as synthetic polymers and urea-denatured proteins.