Direct evidence for alteration of unfolding profile of a helical peptide by far-ultraviolet circular dichroism aromatic side-chain contribution.

Aromatic side-chains are known to contribute to the far-UV circular dichroism (CD) spectra of peptides and proteins. Among other things, this can significantly affect the measured helix propensities of amino acids [Chakrabartty et al., Biochemistry 32 (1993) 5560-5565]. In order to address how interfering side-chain contributions can affect the backbone unfolding transition of a helical peptide, as monitored by [theta;](222) (molar ellipticity at 222 nm), we have studied the unfolding transition of a short designed (alpha-amino isobutyric acid/alanine-based) helical peptide containing an interacting Tyr residue. The guanidinium hydrochloride-induced unfolding of the peptide, as monitored by [theta;](222), showed the presence of a sharp transition superposed over a much broader transition. When the same experiment was performed with a similar peptide that lacked the interacting Tyr residue, the sharp transition disappeared and only the broad transition remained. The sharp transition was assigned to originate from the interacting Tyr side-chain. This demonstrates that conformationally restricted aromatic side-chains that interact with the helical backbone not only can alter the backbone far-UV CD signal, they may also alter the unfolding profiles, monitored by far-UV CD, rendering them unfit for a simple analysis for extracting the appropriate unfolding thermodynamic parameters.