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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-p 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-p interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.Keywords: cation-p interactions; second derivative UV spectra; protein structure; dynamics; peak shift; aromatic amino acids; side chains Supplemental material: see www.proteinscience.org.Dynamic aspects of protein structure have become of increasing interest due to their potential role in protein structure, stability, and function. The structure and motions of polypeptide chains in proteins can be studied by a number of methods, each yielding a different picture of the dynamic structure due to the time scale and magnitude of the motions that can be probed by a particular technique. High resolution pictures are achievable using NMR (Keniry and Carver 2002;Adams et al. 2004), ESR (Poluektov et al. 2003), crystallographic temperature factors (Yuan et al. 2003), isotope exchange monitored by mass spectrometry (Zhang and Smith 1993;Yan et al. 2004) and infrared spectroscopy (Yu et al. 2004). A somewhat lower resolution but unique picture of protein dynamic behavior is provided by using small neutral solutes such as O 2 and acrylamide (Lakowicz et al. 1983;Fasano et al. 2003;Ruiz et al. 2003) Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

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