Glucose-Based Amphiphilic Telomers Designed to Keep Membrane Proteins Soluble in Aqueous Solutions: Synthesis and Physicochemical Characterization

Abstract

A novel class of nonionic amphipols (NAPols) designed to handle membrane proteins in aqueous solutions has been synthesized, and its solution properties have been examined. These were synthesized through free radical cotelomerization of glucose-based hydrophilic and amphiphilic monomers derived from tris(hydroxymethyl)acrylamidomethane using azobisisobutyronitrile as the initiator and thiol as the transfer agent. The molecular weight and the hydrophilic/lipophilic balance of the cotelomers were modulated by varying the thiol/monomers and the hydrophilic monomer/amphiphilic monomer ratios, respectively, and were characterized by 'H NMR, UV, gel permeation chromatography, and Fourier transform infrared spectroscopy. Their physicochemical properties in aqueous solution were studied by dynamic light scattering, aqueous size-exclusion chromatography, analytical ultracentrifugation, and surface-tension measurements. NAPols are highly soluble in water and form, within a large concentration range, well-defined supramolecular assemblies with a diameter of approximately 6-7 nm, a narrow particle size distribution, and an average molecular weight close to 50 x 10(3) g x mol(-1). Varying the hydrophilic/amphiphilic monomer ratio of NAPols in the range of 3.0-4.9, the degree of polymerization in the range of 51-78, and the resulting average molar mass in the range of 20-29 x 10(3) g x mol(-1) has little incidence on their solution properties. Glucose-based NAPols efficiently kept soluble in aqueous solutions two test membrane proteins: bacteriorhodopsin and the transmembrane domain of Escherichia coli's outer membrane protein A.

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“…NS5A-FL/NAPol was prepared as reported in Bazzacco et al (39). NAPols are totally nonionic, amphiphilic polymers synthesized by free-radical polymerization of acrylamide monomers in the presence of a thiol-based transfer agent (40). Homopolymeric NAPols result from polymerization of an amphiphilic monomer carrying two glucose moieties and a single undecyl alkyl chain (41).…”

Folding and stability of integral membrane proteins in amphipols

“…However even for 78 AIDA, folding was successful when the protein was bound to a 79 nickel-bearing column [33]. 80 a-Helical TMPs like BR or KcsA have been folded from dena- 81 tured forms in organic solvent like trifluoroacetic acid or mixtures 82 of formic acid and ethanol [2,11]. Folding of BR was initiated by 83 transferring denatured bacterio-opsin (BO, the apoprotein) first 84 into micelles of the denaturing detergent sodium dodecylsulfate 85 (SDS) and then, usually in the presence of retinal, BR's cofactor, into 86 ''mild'' detergents, mixtures of detergent and lipids, or pure lipids 87 [2-4].…”

“…Heteropolymeric 162 NAPols (Fig. 1C) have been obtained either by cotelomerization 163 of hydrophobic and hydrophilic monomers [82] or by randomly 164 grafting hydrophobic chains onto a glycosylated homotelomer 165 [83]. PC-APols are zwitterionic at neutral and basic pH, cationic 166 at acidic pH.…”

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“…[37] Co-telomerization reactions (Scheme 3) between tris(acetoxymethyl)acrylamidomethane monomer 10 and g-amino propionic acid-derived acrylamide monomer 11, [31] in the presence of telogen 9 as a transfer reagent and azobisisobutyronitrile (AIBN) as a radical initiator, were carried out under an inert atmosphere in refluxing THF. Telomerization was continued until complete consumption of starting monomers (monitored by TLC).…”

“…[37] A study performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) also yielded inconsistent results (not published), probably due to the variable fragmentation behavior of the telomers during the MALDI experiments. This disparity between results observed for synthetic polymers has already been reported in the literature and ascribed to both instrument and sample preparation parameters used for the MALDI-MS analysis.…”