Structural and functional studies of membrane proteins remain a challenge due to difficulties in their handling. In the last decade, several lipoprotein based methods have been proposed for solving exactly this problem in biophysical and functional studies.1The lipoproteins used for this purpose, were derived from naturally occurring protein analogues and in particular, from the amphipathic 243 amino acid long Apolipoprotein A1 (ApoA1), which is the main constituent in high density lipoproteins (HDLs), the carriers of so-called “good” cholesterol.
It is well known that ApoA1 by itself can form discoidal particles when reconstituted with phospholipids,2 and there is accumulating evidence that some peptides that mimic ApoA1 also form discoidal shaped particles when associated with lipids.3 The research groups of Professors Knud J. Jensen and Lise Arleth at the University of Copenhagen, demonstrated that the amphipathic peptide called ‘18A’4 formed peptide nano discs, which could hold membrane protein in a native membrane- like environment.5 The peptides form a double belt around 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid molecules.
In the literature, this peptide is known as ‘18A’ (Ac-18A-NH2). The basic sequence for the synthetic peptide has the following 18 amino acid sequence DWLKAFYDKVAEKLKEAF and is acetylated at the N-terminus and amidated at the C-terminus. The N-terminal acetylation removes a positive charge and intro- duces a carbonyl that has been shown to stabilize the α-helical structure of the peptide.
Figure 1: Peptide nanodiscs that can hold transmembrane proteins.
For these studies, the research groups of Jensen and Arleth required >450 mg of pure peptide ‘18A’ which was synthesized on a Biotage® Initiator+ Alstra™ fully automated microwave peptide synthesizer in a single 30 mL reaction vial.
All materials were obtained from commercial suppliers; Sigma- Aldrich (acetonitrile, formic acid, triethylsilane (TES), hydrazine and dichloromethane (DCM)), Iris Biotech GmbH (Fmoc-amino acids, and N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]- N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), 1-hydroxy-7-azabenzotriazole (HOAt), trifluoroacetic acid (TFA), piperidine and N,N-diisopropylethylamine (DIPEA)) and Rapp Polymere GmbH (TentaGel S Rink Amide resin). Milli-Q (Millipore) water was used for LC-MS analysis.
Nα-9-fluorenylmethoxycarbonyl (Fmoc) amino acids contained the following side-chain protecting groups: tert-butyl (Tyr, Asp and Glu) and tert-butyloxycarbonyl (Lys and Trp).
The ‘18A’ peptide (Ac-18A-NH2) was prepared by Fmoc solid- phase peptide synthesis on a Biotage® Initiator+ Alstra™ microwave peptide synthesizer. The synthesis was carried out on TentaGel S Rink Amide resin (loading 0.25 mmol/g) on a 0.4 mmol scale in a 30 mL reactor vial.
Nα-Fmoc deprotection was performed at room temperature (RT) in two stages by treating the resin with piperidine/DMF (2:3) for 3 min followed by piperidine/DMF (1:4) for 15 minutes. The resin was then washed with NMP (x2), DCM (x1) and again with NMP (x2).
Peptide couplings were performed using 4 eq. of Fmoc-amino acids, 4 eq. of HOAt, 3.9 eq. of HBTU and 7.2 eq. of DIPEA in NMP. A coupling time of 10 minutes at 75 °C was employed and after each coupling step the resin was washed with NMP (x2), DCM (x1) and again with NMP (x2).
After the synthesis was completed, the resin was washed with DCM (x5) and thoroughly dried. The peptide was cleaved from the solid support by treatment with TFA-H2O-TES (95:2:3) for 2 h. The TFA solution was concentrated by nitrogen flow and the peptide was precipitated with cold diethyl ether to yield the crude product. The crude peptide was purified by RP-HPLC (Dionex Ultimate 3000 system) on a preparative 110 Å C18 column (Phenomenex Gemini, 5 µm, 21.2×100 mm) using the following solvent system: solvent A, water containing 0.1% TFA; solvent B, acetonitrile containing 0.1% TFA. Gradient elution (0–5 min: 5% to 25%, 5–32 min: 25% to 60%) was applied at a flow rate of 15 mL min-1. The peptide purity was assessed by LC MS on a Dionex Ultimate 3000 system and the identification was carried out by ESI-MS (MSQ Plus Mass Spectrometer, Thermo). Due to the relatively low solubility of the crude ‘18A’ peptide and the large quantity of final product required, a large number of purification runs were needed.
The ‘18A’ peptide sequence was assembled using SPPS as described above with microwave heating during the coupling steps. The resin was washed and the peptide released from the solid support as described above to afford the desired peptide with a crude purity of 76% and confirmed by ESI-MS (Figure 2), calculated average isotopic composition for C108H160N24O28: 2242.58 Da. Found: m/z 1122.1 [M+2H]2+and 748.3 [M+3H]3+.
Figure 2: RP-HPLC chromatogram and ESI-MS of crude 18A peptide
We have demonstrated the high yield and recovery of the amphipathic peptide ‘18A’ (Ac-18A-NH2) synthesized in a single 30 mL reactor vial. The research groups of Professors Knud J. Jensen and Lise Arleth at the University of Copenhagen, required a large amount of pure peptide ‘18A’ for their structural studies on lipid–peptide particles which form peptide nanodiscs and are a possible solution for the handling of membrane proteins.
The combination of using high concentrations of reagents (0.5M) and fast and precise microwave heating to increase the coupling efficiency using the Biotage® Initiator+ Alstra™ microwave peptide synthesizer, enabled the efficient synthesis of peptide ‘18A’ and simplified the subsequent purification by RP-HPLC despite its relatively low solubility, to afford the desired peptide in 52% yield (468 mg).
Prof. Knud J. Jensen and Dr. Kasper Kildegaard Sørensen at the University of Copenhagen.
Literature number: AN103