Spectroscopic studies reveal conformational flexibility of intrinsically unstructured protein HYPK

Abstract

The chaperone-like huntingtin-interacting protein, HYPK, has unusual biophysical behavior like an intrinsically unstructured protein (IUP). The protein exists as a (pre-) molten globule with ~37% residual structure and shows com- paction in presence of Ca⁺⁺. HYPK contains no intrinsic fluorophore other than a single tyrosine and displays an anomalous fluorescence peak at around 340 nm. The anomalous peak is re- duced to 303 nm by the addition of guanidine hydrochloride and at low pH, concomitant with the emission spectrum of L-tyrosine. At high pH the peak is shifted to ~350 nm with a reduction in intensity. In presence of sodium perchlorate there is no shift in HYPK fluorescence emission peak from ~340 nm suggesting localization of the lone tyrosine residue in helical regions. In CD experiments, however, a shift in local secondary structure is noticed upon perchlorate treatment. Acrylamide quenching experiments at different Ca⁺⁺ concentrations demonstrate that Ca⁺⁺ does not alter the accessibility of the tyro- sine to acrylamide. In the absence of any tryp- tophan contamination, these observations vali- date that, <i>in vitro</i>, HYPK possesses a loosely associated (pre-) molten globule like conforma- tion with the lone tyrosine being situated within an α-helix.