Utility of 5-Cyanotryptophan Fluorescence as a Sensitive Probe of Protein Hydration

Abstract

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.

Do guanidinium and tetrapropylammonium ions specifically interact with aromatic amino acid side chains?

“…Indeed, our CD results (SI Appendix, Figs. S16 and S17) show that although TPA + clusters can induce α-helix formation in an alanine-based peptide, as observed by Dempsey et al (24), they do not increase the α-helicity of a Trp3/Trp CN mutant of the antimicrobial peptide mastoparan-X that is known to form an amphipathic α-helix upon binding to the interfacial region of lipid micelles or membranes (30). These results suggest that unlike membrane-mimic micelles formed by surfactants, a single TPA + cluster is unable to provide a continuous water-hydrophobic interface large enough to host an entire α-helix.…”

“…To help better understand the results obtained with linear and nonlinear IR spectroscopic methods, we also performed fluorescence measurements. It has been shown that the fluorescence quantum yields of Phe CN and Trp CN are sensitive to interactions with water molecules (29,30), with a relation that hydration increases/decreases the fluorescence intensity of Phe CN / Trp CN . Thus, both unnatural amino acids can in principle be used as fluorescence probes to assess how Gdm + and TPA + affect the local hydration status of Phe CN and Trp CN in GF CN G and GW CN G. However, we only carried out fluorescence experiment on GW CN G because Cl − is known to significantly quench the fluorescence of Phe CN (29).…”

“…To achieve this goal, herein we use multiple spectroscopic methods and two unnatural amino acids, p-cyano-phenylalanine (Phe CN ) and 5-cyano-tryptophan (Trp CN ), to directly assess whether protein aromatic side chains can preferentially interact with Gdm + and TPA + ions. Both the C≡N stretching vibration (27,28) and fluorescence quantum yield (29,30) of Phe CN and Trp CN have been shown to be sensitive to their local environment. Thus, the premise of our study is that any specific ion-side chain Significance Ever since the discovery by Franz Hofmeister in 1888 that a series of salts (now commonly referred to as the Hofmeister series) can have different but consistent effects on the solubility and hence stability of proteins, many studies have been devoted to understanding how such effects are achieved.…”

On the positional and orientational order of water and methanol around indole: a study on the microscopic origin of solubility

“…This is consistent with previous neutron diffraction measurements on indole/methanol/water solutions, 9 as well as with infra-red measurements on indole derivatives in solution which have been shown to be sensitive to -OH bonding to the benzene ring motifs. 31,32 From the excess entropy calculations here, this appears to be enthalpically driven rather than entropically driven given that methanol is more ordered compared with water around indole in solution and indole has a much higher solubility in methanol compared with water.…”

Blue fluorescent amino acid for biological spectroscopy and microscopy

“…Many past studies have focused on Trp-based unnatural amino acids, including azatryptophans (5,6,12) and various indole-ring substituted analogs (13)(14)(15)(16), aiming to identify useful biological fluorophores. Whereas some Trp analogs indeed exhibit improved fluorescent properties over Trp, none of them has found broad applications due to certain photophysical limitations.…”