Probing protein structure and dynamics by second‐derivative ultraviolet absorption analysis of cation–π interactions

Abstract We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li + , Na + , and Cs + ) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation–π interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second‐derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N ‐acetylated C ‐ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation–π interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.