All Fmoc-protected amino acids, N,N′-diisopropylcarbodiimide (DIC), OxymaPure® (ethyl-2-cyano-2-(hydroxyimino)acetate), and Fmoc-Lys(Boc)-Wang Tentagel^® resin were purchased from Iris Biotech GmbH (Marktredwitz, Germany). Tentagel^® S RAM was purchased from Rapp Polymere (Tuebingen, Germany). Peptide-synthesis grade N,N′-dimethylformamide (DMF) and acetonitrile (ACN) were purchased from Carlo Erba (Milan, Italy). Dichloromethane (DCM), trifluoroacetic acid (TFA), triisopropylsilane (TIS), and piperidine were purchased from Sigma-Aldrich (Milan, Italy). 2,2,2-trifluoroethanol (TFE) was purchased from Alfa Aesar (Kandel, Germany). Myelin Basic Protein (MBP) was purchased from Merck (Milan, Italy).

Peptides 1-6 were synthesized in solid-phase using a microwave-assisted protocol (MW-SPPS) on a Liberty Blue™ automated peptide synthesizer (CEM Corporation, Matthews, NC, United States), following the Fmoc/tBu strategy as previously described (Rizzolo et al., 2011; Pandey et al., 2013; Nuti et al., 2020). Tentagel® S RAM resin (loading 0.23 mmol/g) was used for the synthesis of peptides 1, 5, and 6. Fmoc-Lys(Boc)-Wang Tentagel® resin (loading 0.23 mmol/g) was used for the synthesis of peptides 2, 3, and 4. Fmoc-deprotections were performed with a solution of 20% (v/v) piperidine in DMF (2 M). Peptide assembly was performed by repeating the standard MW-SPPS coupling cycle for each amino acid, using Fmoc-protected amino acids (2.5 equiv, 0.4 M in DMF), OxymaPure® (2.5 equiv, 1 M in DMF), and DIC (2.5 equiv, 3 M in DMF). Uncertain peptide coupling steps were checked by the ninhydrin test described by Kaiser (Kaiser et al., 1970), or micro-cleavages performed with a microwave apparatus CEM Discover™ single-mode MW reactor (CEM Corporation, Matthews, NC, United States). Resins with peptides 2-4 were N-terminal acetylated before cleavage, using twice Ac₂O in a DMF solution for 10 min at room temperature. Final cleavage and side-chain deprotections were performed using a mixture of TFA/TIS/H₂O (95:2.5:2.5, v/v/v) at room temperature. After 2.5 h each resin was filtered off and the solution was concentrated flushing with N₂. Each peptide was precipitated from cold Et₂O, centrifuged, and lyophilized.

The crude peptides were purified by Reverse-Phase Flash Liquid Chromatography (RP-HPLC) on an Isolera One Flash Chromatography (Biotage, Uppsala, Sweden) using a SNAP Ultra C18 column (25 g) at 20 ml/min. Eluent systems: 0.1% (v/v) TFA in H₂O (A), 0.1% (v/v) TFA in ACN (B) (elution gradient reported in Supplementary Table S1). The second step of purification of the peptides was performed by semipreparative RP-HPLC on a Waters instrument (Separation Module 2695, detector diode array 2996) using a Sepax Bio-C18 column (Sepax Technologies, Newark, United States) (5 μM, 250 mm × 10 mm), at 4 ml/min using the solvent systems A (0.1% TFA in H₂O) and B (0.1% TFA in ACN). Characterization of the peptides was performed by analytical HPLC using a Waters ACQUITY HPLC coupled to a single quadrupole ESI-MS (Waters® ZQ Detector, Waters Milford, MA, United States) supplied with a BEH C18 column (1.7 μm, 2.1 mm × 50 mm) at 35°C, 0.6 ml/min with the solvent systems A (0.1% TFA in H₂O) and B (0.1% TFA in ACN). Gradient elution was performed with a flow of 0.6 ml/min and started at 10% B, with a linear increase to 90% B in 5 min. The analytical data are reported in the Supplementary Material (Supplementary Table S1 and Supplementary Figures S1–S6).

CD spectra were recorded on JASCO J-815 with increasing temperature from 10 to 60°C in increments of 5°C between λ = 270 and 185 nm. The CD measurements were carried out in H₂O, a mixture of H₂O:TFE (50:50, v:v) and PBS (phosphate buffered saline, pH 7.4, containing: sodium chloride, potassium chloride, sodium phosphate dibasic, potassium phosphate monobasic). The spectra were registered with the following parameters: 0.2 nm resolution, 1.0 nm bandwidth, 20 mdeg sensitivity, 0.25 s response, 100 nm min⁻¹ scanning speed, 5 scans, and 0.02 cm cuvette path length. The CD spectra of solvents were recorded and subtracted from the raw data. The spectra were corrected by a baseline that was measured with the identical solvent in the same cell. The CD intensity is given as mean residue molar ellipticity (θ) [deg × cm² × dmol⁻¹].

The samples for NMR spectroscopy were prepared by dissolving the appropriate amount of peptide in 50 mM potassium phosphate buffer (pH 6.5), 10% D₂O, and 100 mM DPC-d₃₈. NMR spectra were recorded on a Varian INOVA 600 MHz spectrometer equipped with a z-gradient 5 mm triple-resonance probe head. Spectra were recorded at a temperature of 25°C. The spectra were calibrated relative to TSP (0.00 ppm) as internal standard. One-dimensional (1D) NMR spectra were recorded in the Fourier mode with quadrature detection. The water signal was suppressed by gradient echo (Hwang and Shaka, 1995). 2D DQF-COSY (Piantini et al., 1982; Marion and Wüthrich, 1983), TOCSY (Braunschweiler and Ernst, 1983), and NOESY (Jenner et al., 1979) spectra were recorded in the phase-sensitive mode. Data block sizes were 2048 addresses in T2 and 512 equidistant T1 values. Before Fourier transformation, the time domain data matrices were multiplied by shifted sin2 functions in both dimensions. A mixing time of 70 ms was used for the TOCSY experiments. NOESY experiments were run with mixing time of 100 ms.

Many publications indicate the presence of an immunodominant sequence in a specific region of MBP. One report described the sequence MBP (83–101) as a potential immunodominant T-cell epitope restricted to HLA-DR2b (DRB1*15:01) in MS (Ota et al., 1990), while others reported that MBP (89–101) peptide has two registers for binding to DR2a and DR2b (Kim et al., 2014). Other authors identified MBP (87–106) (Martin et al., 1990), MBP (83–101) (Hansen et al., 2007), and other overlapping sequences as T-cell epitopes inside the MBP protein. Importantly, these MBP regions match with B-cell epitopes previously described (Lolli et al., 2005; Warren et al., 2006; Mameli et al., 2014).

With the idea in mind to cover the whole immunodominant region of MBP, we synthesized and tested the peptide MBP (81–106) (1). Moreover, considering that the sensitivity of the peptide-based ELISA might be limited when short antigen sequences are used, we designed the elongated sequence MBP (76–116) (2), in order to achieve better antigen exposition in the solid-phase conditions of the ELISA (Nuti et al., 2020). With the aim of clarifying the amino-acid residues specifically involved in the B-cell epitope recognition, we divided the whole sequence of MBP (76–116) (2) into two fragments: MBP (76–96) (3) and MBP (97–116) (4). Amino- and carboxy-termini of peptides were acetylated and amidated, respectively, in order to remove free terminal charges, which are not present in the native protein sequence and may interfere with antibody recognition (Van Regenmortel, 2001). Finally, we also synthesized the shorter sequences MBP (81–92) (5) and MBP (99–106) (6), to be tested in competitive ELISA.

A preliminary screening of the conformational preferences of peptides MBP (81–106) (1) and MBP (76–116) (2) indicate that these peptides appear randomly structured in water solution (Figure 3A; Supplementary Figure S14). We subsequently studied the conformational behavior of these peptides in H₂O:TFE (50:50, v:v), a solvent mixture known to act as stabilizing agent (Campagna et al., 1998). Moreover, the use of fluoroalcohol provides additional stability by removing the water molecules from the surroundings of the peptides (Khandelwal et al., 1999). The spectra obtained in the solvent mixture displayed two negative bands at 222 and 208 nm (n→π* and π→π* transitions) and one positive band at 190 nm (π→π* transition) (Figure 3B), characteristic for helical structures according to the literature (Bradley et al., 1990; Bates et al., 2004; Ahmed et al., 2012; Miles et al., 2022). Indeed, predictions based on amino acid composition of these peptides indicate that they show a tendency to form helical structures (Yang et al., 1997; Bates et al., 2004; Ahmed et al., 2012). Moreover, it may be assumed that there were no spectral differences of secondary peptide structure at various temperatures (Supplementary Figure S14, S15). Interestingly, the shorter peptide MBP (81–106) (1) displays higher tendency to adopt an helical conformation, as compared to the longer analogue MBP (76–116) (2) (% helix 14 vs. 3) (Figure 3B) (Sommese et al., 2010). Apparently, the elongation at N- and C-termini of the peptide causes the disability in forming the helix. Furthermore, a different agent stabilizing the helix motif in the structure of MBP (81–106) (1) is the presence at the C- and N-termini of positively and negatively charged amino acids able to form salt bridges (Forood et al., 1993). At variance, the longer peptide MBP (76–116) (2) includes many positively charged residues, i.e., arginine residues that produce a destabilized effect on helix conformation by electrostatic repulsion (Sitkoff et al., 1994).

We subsequently studied the conformational preferences of peptides MBP(81–106) (1) and MBP (76–116) (2) in PBS, which is the most frequently used solvent to maintain physiological pH (White, 2002). These data indicate that the peptides present random coil structures (Figure 4). In fact, the obtained spectra displayed a single band with negative ellipticity at approximately 200 nm characteristic for unordered peptides. However, peptide MBP (81–106) (1) presents a more ordered structure, as can be inferred by the small but significant shift at higher wavelength of the minimum of MBP (81–106) (1) compared to MBP (76–116) (2) (Muruganandam et al., 2013; Lanthier et al., 2014). Moreover, we can assume there were no differences of secondary structure at various temperatures for MBP(81–106) (1) (Figure 5). In contrast, increasing the temperature of MBP (76–116) (2) led to a shift of the minimum to lower wavelength and a strong decrease of the signal intensity (Figure 6). This result can derive from a temperature-induced peptide self-aggregation, likely in beta structures (minimum at about 205 nm). The partial aggregation can account for the reduced ability of MBP (76–116) (2) to interact with the IgM antibody.

Conformational behavior of peptides MBP (81–106) (1) and MBP (76–116) (2) was studied in DPC micelle (100 mm) by solution NMR spectroscopy. Almost complete ¹H NMR assignments were not was obtained for peptide 1 while some residues of 2 could not be assigned due to strong signals overlapping (Supplementary Tables S2, S3). From chemical shift and NOE interaction patterns (Supplementary Tables S2, S3; Supplementary Figure S16), the two peptides have stable helical structure along residues 87–96, while the remaining regions did not show diagnostic parameters of any secondary structure. They are henceforth in random coil conformation. These results are in agreement with those reported in the literature (Ahmed et al., 2012) about a 36-residue peptide fragment of murine MBP corresponding to residues 76–111. Comparing the chemical shift values peptides MBP (81–106) (1) and MBP (76–116) (2), which are not significantly different, the helical structure turns out to have the same stability regardless of the sequence length. Superposition of chosen regions of the NOESY spectra of the two peptides (Supplementary Figure S16) gives a visual confirmation of this result.