Tryptophan (Trp) fluorescence has been widely used to interrogate the structure, dynamics and function of proteins. In particular, it provides a convenient and site-specific means to probe a protein’s hydration status and dynamics. Herein, we show that a tryptophan analog, 5-cyanotryptophan (TrpCN), can also be used for this purpose, but with the benefit of enhanced sensitivity to hydration. This conclusion is reached based on measurements of the static and time-resolved fluorescence properties of 5-cyanoindole, TrpCN, and TrpCN–containing peptides in different solvents, which indicate that upon dehydration the fluorescence quantum yield (QY) and lifetime (τF) of TrpCN undergo a much greater change in comparison to those of Trp. For example, in H2O the QY of TrpCN is less than 0.01, which increases to 0.11 in 1,4-dioxane. Consistently, the fluorescence decay kinetics of TrpCN in H2O are dominated by a 0.4 ns component, whereas in 1,4-dioxane the kinetics are dominated by a 6.0 ns component. The versatile utility of TrpCN as a sensitive fluorescence reporter is further demonstrated in three applications, where we used it (1) to probe the solvent property of a binary mixture consisting of dimethyl sulfoxide and H2O, (2) to monitor the binding interaction of an antimicrobial peptide with lipid membranes, and (3) to differentiate two differently hydrated environments in a folded protein.

Several cyanotryptophans have been shown to be useful biological fluorophores. However, how their fluorescence lifetimes vary with solvent has not been examined. In this regard, herein we measure the fluorescence decay kinetics as well as the absorption and emission spectra of six cyanoindoles in different solvents. In particular, we find, among other results, that only 4-cyanoindole affords a long fluorescence lifetime and hence high quantum yield in H2O. Therefore, our measurements provide not only a guide for choosing which cyanotryptophan to use in practice but also data for computational modeling of the substitution effect on the electronic transitions of indole.

Many ions are known to affect the activity, stability, and structural integrity of proteins. Although this effect can be generally attributed to ion-induced changes in forces that govern protein folding, delineating the underlying mechanism of action still remains challenging because it requires assessment of all relevant interactions, such as ion-protein, ion-water, and ion-ion interactions. Herein, we use two unnatural aromatic amino acids and several spectroscopic techniques to examine whether guanidinium chloride, one of the most commonly used protein denaturants, and tetrapropylammonium chloride can specifically interact with aromatic side chains. Our results show that tetrapropylammonium, but not guanidinium, can preferentially accumulate around aromatic residues and that tetrapropylammonium undergoes a transition at ∼1.3 M to form aggregates. We find that similar to ionic micelles, on one hand, such aggregates can disrupt native hydrophobic interactions, and on the other hand, they can promote α-helix formation in certain peptides.T he stability of a protein, or more precisely, the free energy difference between its folded and unfolded states, can be modulated by various solution properties. For example, addition of another solute to a protein solution can result in either a decrease or an increase in this protein's stability (1-3). The most noticeable example in this regard is the Hofmeister series (4-6), a group of ions that are ranked based on their protein-denaturing abilities. Among this series, the guanidinium ion (Gdm + ) is the most widely used chemical agent in protein denaturation due to its strong destabilizing effect and high solubility in water. Consequently, its mechanism of action has been subjected to extensive studies (7)(8)(9)(10)(11)(12)(13)(14). Although different interpretations have been put forth (15-17), the generally accepted notion is that Gdm + denatures a protein by preferentially interacting with its peptide groups (7,11,18), including certain side chains (11,(19)(20)(21)(22). In particular, it has been hypothesized that Gdm + , which exists in aqueous solution as a rigid, flat object (12,18,19), can engage in stacking interactions with amino acids consisting of planar side chains, such as arginine (Arg) (19), asparagine (Asn) (11,20), glutamine (Gln) (11,20), and aromatic residues (20)(21)(22). However, to the best of our knowledge, the only experimental evidence that supports this hypothesis comes from crystallographic data (20)(21)(22), which show that the guanidinium group of Arg is more frequently found to be stacked against the side chain of tyrosine (Tyr) or tryptophan (Trp) in proteins. Therefore, additional experimental studies that can directly probe such stack interactions in solution are needed.Another ion in the Hofmeister series that has a similar proteindenaturing capability to Gdm + is tetrapropylammonium (TPA + ) (23). However, a recent study by Dempsey et al. (24) found that although TPA + , like Gdm + , can denature a tryptophan (Trp) zipper β-hairpin (i.e....

Considerable efforts have been devoted to the development of spectroscopic probes that are sensitive to water and can be used to monitor, for example, biological and chemical processes involving dehydration or hydration. Continuing this line of research, herein we show that 7-cyanoindole can serve as a sensitive fluorescence probe of hydration as its fluorescence properties, including intensity, peak wavelength and lifetime, depend on the amount of water in nine water-organic solvent mixtures. Our results indicate that 7-cyanoindole is not only able to reveal the underlying microheterogeneity of these binary solvent systems, but also offers distinct advantages. These include: (1) its fluorescence intensity increases more than ten times upon going from a hydrated to a dehydrated environment; (2) its peak wavelength shifts as much as 35 nm upon dehydration; (3) its single-exponential fluorescence decay lifetime increases from 2.0 ns in water to 8-16 ns in water-organic binary mixtures, making it viable to distinguish between differently hydrated environments via fluorescence lifetime measurements; and (4) its absorption spectrum is significantly red-shifted from that of indole, making selective excitation of its fluorescence possible in the presence of naturally occurring amino-acid fluorophores. Moreover, we find that for seven binary mixtures the fluorescence lifetimes of 7-cyanoindole measured at solvent compositions where maximum microheterogeneity occurs correlate linearly with the peak wavenumbers of its fluorescence spectra obtained in the respective pure organic solvents. This suggests that the microheterogeneities of these binary mixtures bear certain similarity, a phenomenon that warrants further investigation.