Harmonic Analysis of the Fluorescence Response of Bimane Adducts of Colicin E1 at Helices 6, 7, and 10

Abstract

Background: Colicin E1 forms a voltage-dependent channel in the cytoplasmic membrane of target E. coli cells. Results: Fluorescence analyses reveal that helices 6, 7, and 10 in the membrane-bound colicin E1 are separate amphipathic ␣-helices with different periodicities. Conclusion:A new model of the closed channel of colicin E1 has been developed. Significance: This study provides new data on the structure of the umbrella model for the channel-forming colicins.

Site-Directed Fluorescence Approaches for Dynamic Structural Biology of Membrane Peptides and Proteins

“…A plot of emission maximum as a function of dielectric constant is shown for the widely used intrinsic fluorophore of a protein, Trp (indole as the fluorescing group) and for the popular extrinsic probes for site-directed fluorescence, NBD and bimane. Data are adapted from Sun and Song (1977) (indole); Fery-Forgues et al (1993) (NBD); Ho et al (2013) (bimane) and plotted. See text for details.…”

“…This is particularly true for membrane peptides and proteins for which there is no high-resolution structure available or when the structural information is limited. This is reflected in the extensive use of SDFL methods to study changes in conformational dynamics in different classes of membrane proteins like pore-forming peptides and proteins (Nagahama et al, 2002; Parker and Feil, 2005; Raghuraman and Chattopadhyay, 2007a; Haldar et al, 2008; Ho et al, 2013), GPCR (Yao et al, 2006; Daggett and Sakmar, 2011; Dekel et al, 2012; Alexiev and Farrens, 2014), potassium channels (Cha and Bezanilla, 1997, 1998; Cha et al, 1999; Raghuraman et al, 2014), inward-rectifying potassium channels (Wang et al, 2016, 2018, 2019), mechanosensitive ion channels (Wang et al, 2014; Martinac, 2017), ligand-gated ion channels (Sasmal and Lu, 2014), membrane transporters (Liu and Sharom, 1996; Verhalen et al, 2012; Terry et al, 2018), and intrinsically disordered proteins (Ferreon et al, 2009). Therefore, the wide applicability of SDFL approaches to study diverse systems makes fluorescence a sophisticated yet reliable technique for ensemble and single molecule measurements in both in vitro and in vivo .…”

Resolving the 3D spatial orientation of helix I in the closed state of the colicin E1 channel domain by FRET. Insights into the integration mechanism

“…The single-Trp variants of ColE1 c showed a more homogeneous fluorescence decay in the membrane-bound forms than in the soluble forms (shaded rows in both panels of Table 1), which may correspond to a more similar environment around each emitter in the former than in the latter condition [32]. For the membranebound form, the bimane acceptor at the 354 site produces a larger effect on Trp fluorescence than at the two other positions (347 and 361) by reducing either the average lifetime of the Trp424 signal by the largest amount, and the average lifetimes of the Trp460 and Trp495 signals by the least amount.…”

“…Protein purities were assessed by SDS-PAGE and protein concentrations were determined by spectroscopy at A 280 , using an extinction coefficient (ε) of 29910 M À1 cm À1 for WT protein (ε ¼ 17210 M À1 cm À1 for single Trp variant protein) [35]. Purified single-Trp, single-Cys variants were labeled with the small fluorophore monobromobimane (mBBr, 271.11 g/mol) (Molecular Probes, Eugene, USA) at a 20:1 M ratio (probe:protein), and the labeling efficiency was determined as previously described [32]. LUVs were prepared from 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glyerco-3-[phospho-rac-(1-glycerol)] vesicles at a 60:40 M ratio (Avanti Polar Lipids).…”

“…To further test the proposed quasi-circular arrangement model, our lab used the system developed by Schultz [30] to incorporate coumarin into the ColE1 channel domain to act as an intrinsic FRET donor with DABMI-labeled Cys residues as the acceptor [31]. This approach led to the development of a circular arrangement model with helices IeVII (H 1 eH 7 ) arranged in a clockwise direction from the extracellular side around the central transmembrane hairpin formed by H 8 and H 9 [32].…”