Donor–Donor Energy‐Migration Measurements of Dimeric DsbC Labeled at Its N‐Terminal Amines with Fluorescent Probes: A Study of Protein Unfolding

Abstract

Fluorescence resonance energy transfer (FRET) is a powerful technique for the determination of distances between two fluorophores. The overall geometry of protein structures [1][2][3][4] and the conformational changes of a molecule under different conditions can be studied by this method if appropriate sites of the molecule are labeled with fluorescence donor and acceptor probes. Nevertheless, it is rather difficult to specifically introduce two different fluorophore groups into one molecule, [2] especially into a homodimeric biomacromolecule that has two identical reactive sites. Different from the conventional FRET technique, donor-donor energy migration (DDEM) takes advantage of certain fluorescence probes that display an overlap of their absorption and emission spectra and are therefore able to transfer energy between themselves.[2-4] Energy transfer in this case is a reversible process and can be measured through analysis of the timeresolved depolarization of the fluorescence emission (as donor-donor energy migration results in additional depolarization). As only one type of probe is required, DDEM simplifies greatly not only the labeling operation but also the theoretical analysis and the time-resolved measurements and has been widely used to study the steady-state conformational changes of biomacromolecules.DsbC (1), a member of the Dsb family in the periplasm of Gram-negative bacteria, is a thiol-protein oxidoreductase that displays molecular chaperone activity. [5][6][7] The DsbC molecule is a V-shaped homodimer consisting of two 23.4-kDa subunits.[8] Each subunit is composed of a C-terminal thiore-

Progress in Spectroscopic Probes with Cleavable Active Bonds

“…However, energy transfer can sometimes take place between the two chemically identical donors, if such a donor molecule has a rather small Stokes shift. This is the so-called homo-FRET, also called donor-donor energy migration (DDEM) [19][20][21]. Because the efficiency of energy transfer depends upon the distance, these fluorescence techniques have found utility in the measurement of the distances between the two interacting fluorophores in macromolecules, especially biomacromolecules.…”

N-Terminal Specific Fluorescence Labeling and its Use in Local Structure Analysis of Proteins (Invited Review)

“…(5) depicts the mechanism of transamination [35][36][37]: the N-terminal amino group of a protein can be converted into a reactive carbonyl group, which can then react with hydrazide-containing fluorophores with no cross-reactivity (Fig. (6)) [17][18][19]. Because the intermediate in transamination reaction involves the participation of the adjacent peptide bond, only the N-terminal amino group is converted, while the internal amino groups ( -amino groups) on lysine residues are not modified [35][36][37].…”

“…Because the intermediate in transamination reaction involves the participation of the adjacent peptide bond, only the N-terminal amino group is converted, while the internal amino groups ( -amino groups) on lysine residues are not modified [35][36][37]. In our recent studies, we have demonstrated this transamination reaction combined with fluorescence donor-donor energy migration to trace the unfolding behavior of a dimeric protein, and combined with a designed polarity-sensitive fluorescent probe to detect the polarity change of the N-terminal domain of proteins [17][18][19].…”

“…In the present contribution, we will make an effort to summarize the development of N-terminal specific labeling and fluorescence probes in local structure analysis of proteins, including some results of our recent studies onlactalbumin, -lactoglobulin, and a dimeric protein of DsbC [17][18][19]. It is our wish that this paper may provide help to those who are interested in the growing research field.…”

Energy migration within BODIPY dimer studied by single molecule spectroscopy

“…In our previous work [7], we employed donor-donor energy migration method with two identical BODIPY dyes as probes to determine the unfolding of a protein DsbC. In this paper, we employ the single molecule technique to investigate the optical behavior of a synthesized BODIPY 650 dimer.…”