Thioamides as Fluorescence Quenching Probes: Minimalist Chromophores To Monitor Protein Dynamics

Abstract

Decreasing the size of spectroscopic probes can afford higher-resolution structural information from fluorescence experiments. Therefore, we have developed p-cyanophenylalanine (Cnf) and backbone thioamides as a fluorophore/quencher pair. Through the examination of a series of thiopeptides, we have determined the working distance for this pair to be 8-30 Å. We have also carried out a proof-of-principle protein-folding experiment in which a Cnf/thioamide-labeled version of villin headpiece HP35 was thermally unfolded while the Cnf/thioamide distance was monitored by fluorescence. For a given protein, thioamide substitutions could be used to track motions with a much greater number of measurements than for current fluorescence probes, providing a dense array of data with which to model conformational changes.

Thioamide Quenching of Fluorescent Probes through Photoinduced Electron Transfer: Mechanistic Studies and Applications

“…26,49 These experiments used p- cyanophenylalanine (Cnf) and thioamide labels to track the distances between pairs of residues in the 8 to 30 Å range over which this FRET probe pair is useful. 27 Our studies complemented similar studies by Deniz and coworkers using longer range FRET pairs. 50 We can use Fam/thioamide pairs to observe short distance interactions that are not resolvable by Cnf/thioamide pairs.…”

“…25,27 The small size of these fluorophores and the relative ease with which they can be genetically incorporated into a protein make them useful tools for a number of applications in studying protein folding or protein-protein interactions. However, the photophysical properties of these probes, particularly their high energy (i.e., short wavelength) excitation energies and low extinction coefficients, prevent them from being used to study protein dynamics in vivo or in single molecule studies.…”

Molecular Dynamics Insight into the Diverse Thermodynamic Behavior of a Beta‐Hairpin Peptide

“…Recently, a minimalist chromophore, backbone thioamide, has been developed to form an intrinsic fluorophore/quencher pair. 52 This technique will make the kind of distance changes that we talk about here more of a reality. In our sample b-hairpin with 15 peptide bonds, there are 7 peptide-bond pairs [see Fig.…”

Sensing pH via p-cyanophenylalanine fluorescence: Application to determine peptide pKa and membrane penetration kinetics

“…For example, dehydration leads to a significant decrease in the fluorescence intensity of Phe CN , thus making it a useful probe of processes that involve exclusion of water, such as protein folding [5,6], binding [7,8], aggregation [9–11], and interaction with membranes [12–15]. In addition, the fluorescence quantum yield of Phe CN can be modulated by various metal ions [16] and several amino acid sidechains [17–21]. In particular, its fluorescence can be quenched by a nearby tryptophan (Trp) residue via the mechanism of fluorescence resonance energy transfer (FRET) [22–24].…”