Aromatic Cluster Sensor of Protein Folding: Near-UV Electronic Circular Dichroism Bands Assigned to Fold Compactness.

Both far- and near-UV electronic circular dichroism (ECD) spectra have bands sensitive to thermal unfolding of Trp and Tyr residues containing proteins. Beside spectral changes at 222 nm reporting secondary structural variations (far-UV range), L-b bands (near-UV range) are applicable as 3D-fold sensors of protein's core structure. In this study we show that both L-b(Tyr) and L-b(Trp) ECD bands could be used as sensors of fold compactness. ECD is a relative method and thus requires NMR referencing and cross-validation, also provided here. The ensemble of 204 ECD spectra of Trp-cage miniproteins is analysed as a training set for " calibrating" Trp$ Tyr folded systems of known NMR structure. While in the far-UV ECD spectra changes are linear as a function of the temperature, near-UV ECD data indicate a non-linear and thus, cooperative unfolding mechanism of these proteins. Ensemble of ECD spectra deconvoluted gives both conformational weights and insight to a protein folding <-> unfolding mechanism. We found that the L-b(293) band is reporting on the 3D-structure compactness. In addition, the pure nearUV ECD spectrum of the unfolded state is described here for the first time. Thus, ECD folding information now validated can be applied with confidence in a large thermal window (5 <= T <= 85 degrees C) compared to NMR for studying the unfolding of Trp <-> Tyr residue pairs. In conclusion, folding propensities of important proteins (RNA polymerase II, ubiquitin protein ligase, tryptase-inhibitor etc.) can now be analysed with higher confidence.