Site-Directed Fluorescence Labeling of a Membrane Protein with BADAN: Probing Protein Topology and Local Environment

Abstract

The work presented here describes a new and simple method based on site-directed fluorescence labeling using the BADAN label that permits the examination of protein-lipid interactions in great detail. We applied this technique to a membrane-embedded, mainly alpha-helical reference protein, the M13 major coat protein. Using a high-throughput approach, 40 site-specific cysteine mutants were prepared of the 50-residues long protein. The steady-state fluorescence spectra were analyzed using a three-component spectral model that enabled the separation of Stokes shift contributions from water and internal label dynamics, and protein topology. We found that most of the fluorescence originated from BADAN labels that were hydrogen-bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method revealed the embedment and topology of the labeled protein in the membrane bilayer under various conditions of headgroup charge and lipid chain length, as well as key characteristics of the membrane such as hydration level and local polarity, provided by the local dielectric constant.

Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin

“…The peak position in the RM sample is initially red shifted compared to that of the solution phase sample, and with the addition of osmolytes, reaches the same end point as the solution phase sample. The red shift is consistent with a slight enhancement of water exposure for the protein bound probe [44]. This conclusion is supported by our observation (not shown) that for Hb-Bd in solution and in a sol–gel, the initial peak position shifts from 470 nm to 527 nm and 506 nm, respectively when denaturants are added (either 6 M urea or 5 M GdHCl).…”

“…Badan can be covalently bound to a thiol group in peptides or proteins through a reaction with the bromoacetyl moiety of the Badan molecule [43]. The emission maximum is strongly correlated to the polarity and the dielectric relaxation properties of the surrounding environment of the probe [44–46]. In the present study Badan is covalently attached to the thiol on glutathione (GSH), the reactive Cys beta 93 sulfydryls of HbA and the reactive thiol mutagenically introduced into myoglobin (S3C).…”

Spectral properties of BADAN in solutions with different polarities

“…Fluorescent solvatochromic dye BADAN (6-bromo-acetyl-2-dimethylamino-naphtalene) ( Figure 1) is used for studying of the phase transitions in biological membranes, protein conformational changes induced by different factors, proteins interactions with their partners and ligands [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]. Furthermore, fluorescence of this dye is used as a recorded signal in biosensor systems [16][17][18][19][20][21].…”

Increased Structural Flexibility at the Active Site of a Fluorophore-conjugated β-Lactamase Distinctively Impacts Its Binding toward Diverse Cephalosporin Antibiotics

“…For our design we decided to use the naphthalene-type fluorophore molecules because of their exceptional solvachromatic properties that can reflect the polarity of the local environmental at the molecule level, as well as their relative small size comparable to the amino acid, tryptophan, to allow them to be positioned close to the protein structure (7)(8)(9)(10)(11)(12)(13). This fluorophore has been applied to probe the structural dynamics of membrane lipids (11) and ion channels (12), and to detect the binding of peptide ligands to SH2 domains (9) and Class II MHC proteins (13).…”