Rational design of novel high molecular weight solubilization surfactants for membrane proteins from the peptide gemini surfactants (PG-surfactants)

Design of PG-Surfactants Bearing Polyacrylamide Polymer Chain to Solubilize Membrane Proteins in a Surfactant-Free Buffer

“…The fluorescence spectral peak at 720 nm, derived from the red-chlorophyll state, was retained To characterize the conjugated structure and properties of the soluble state of PSI with DKDKC 12 K-PA n , we first analyzed the photophysical properties by UV-vis and fluorescence spectroscopy (Figure 4). As shown in Figure 4, all conjugates with DKDKC 12 K-PA 5 , DKDKC 12 K-PA 7 , and DKDKC 12 K-PA 18 showed typical absorption peaks of PSI at 423 and 680 nm in Figure 4a, similar to the case of solubilizing in a buffer with 0.1 wt% β-DDM (control of the native state) [20,21]. On the other hand, an increase in baseline, less than 600 nm region was also observed, which was different from that of solubilizing in a buffer with 0.1 wt% DKDKC 12 K-PA n , especially for the conjugates with DKDKC 12 K-PA 5 and DKDKC 12 K-PA 7 (Figure 2a).…”

“…The RAFT initiator containing the dithiopyridyl (DTP) group, BSTP pyridyl disulfide, was synthesized according to previous study [27]. PSI derived from T. vulcanus was prepared similar to the previous study [21]. Unless otherwise stated, other chemicals and reagents were obtained commercially and used without further purification.…”

“…In higher plants and cyanobacteria, it exists in thylakoid membranes and participates in the reductive side light reaction. Because surfactants have unique photophysical properties derived from the hierarchical supramolecular organization of these components, this protein could be used as a probe to evaluate impacts of surfactants on protein structure and functions from impacts on their photophysical properties [20,21]. The PSI samples solubilized with the buffer K2 (40 mM HEPES−NaOH (pH 7.8), 100 mM NaCl, 15 mM CaCl 2 , and 15 mM MgCl 2 ) containing 0.1 wt% DKDKC 12 K-PA n were prepared by the surfactant-exchange method as previously reported [20].…”

“…Meanwhile, we recently studied Gemini-type peptide surfactants, PG-surfactants [19][20][21][22][23]. The basic molecular scaffold of PG-surfactants consists of three constituents: the linker peptide from to 3-5 residues (X), two alkylamidomethyl-modified Cys residues at both sides of the linker peptide, and the peripheral peptides (Y and Z) at the N-and C-terminal sides of the alkylamidomethyl-modified Cys residues (Figure 1a).…”

“…From a set of screenings on hydrophilic peptide sequences at X, Y, and Z, we found that two PG-surfactants, DKDKC 12 K and DKDKC 12 D, were able to function as solubilization surfactants for membrane proteins [20]. Furthermore, by the tandem connection of the -Cys(C 12 )-Asp-Lys-Asp-Lys-Cys(C 12 )-units in DKDKC 12 K and DKDKC 12 D via flexible (Gly) 4 linkers in one molecule, we succeeded in designing the high molecular weight (MW) solubilization surfactants, Bis-D 3 -DKDKC 12 (MW~3 kDa), Bis-K 3 -DKDKC 12 (MW~3 kDa), and Tris-D 3 -DKDKC 12 (MW 4.3 kDa) [21]. Interestingly, an increase in Mw enhanced the affinity between membrane proteins and surfactants, thereby allowing effective solubilization, even for lower concentration ranges (<0.0005 wt%).…”