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. 2026 Sep 17. Online ahead of print. doi: 10.1039/d6cb00176a

Semi-synthesis of the C-terminal region of PfRALP1: an alternative strategy for producing a hard-to-express merozoite protein

Akash Narayan a, Sreejith Raran-Kurussi a, Mrinal K Bhattacharyya b, Kalyaneswar Mandal a,✉
PMCID: PMC13617758  PMID: 42807854

Abstract

Plasmodium falciparum rhoptry-associated leucine zipper-like protein 1 (PfRALP1) is an indispensable protein involved in merozoite invasion of human red blood cells (RBCs), leading to the deadly malaria disease. PfRALP1 is stored in the rhoptry neck of merozoites and translocated to the moving junction during invasion and is therefore regarded as a moving junction protein. The C-terminal region of the full-length PfRALP1 (PfRALP1-C1) plays a crucial role in binding to an unidentified receptor protein on erythrocytes during merozoite invasion. PfRALP1 also elicits immunogenicity in P. falciparum-infected asymptomatic and semi-immune adults. Successful recombinant expression of PfRALP1-C1 was demonstrated using a wheat germ cell-free extract system. However, obtaining pure recombinant PfRALP1 in other heterologous systems has not yet been explored. We faced significant challenges in obtaining PfRALP1-C1 in pure form in reasonable quantities through overexpression in E. coli. To address this issue, we introduce a semi-synthetic approach to obtain functional PfRALP1-C1. Our findings include an efficient three-segment native chemical ligation of two chemically synthesized peptide segments and a recombinantly expressed polypeptide. The circular dichroism data for the semi-synthetic protein are consistent with the predicted secondary structure, which consists of intrinsically disordered regions and alpha helices. The semi-synthetic PfRALP1-C1 retains the binding epitope and interacts with the unknown RBC receptor, as it competitively inhibits the invasion of RBCs by merozoites. Thus the semi-synthetic method described here offers a promising strategy for producing this difficult-to-express antigenic protein PfRALP1-C1, with potential for large-scale production and downstream biological and therapeutic applications.

Semi-synthetic PfRALP1-C1, the C-terminal region of the indispensable PfRALP1 protein, competitively blocks merozoite invasion of RBCs. This hard-to-express malaria antigen was produced via 3-segment native chemical ligation.graphic file with name d6cb00176a-ga.webp

Introduction

Malaria continues to be a major global health threat to the human population. The malaria-causing parasites cycle between two evolutionarily distinct hosts, female Anopheles mosquitoes (invertebrates) and humans (vertebrates), complicating efforts to control their spread. Among all malaria parasites that infect humans, Plasmodium falciparum accounts for more than 90% of deaths.1,2 This high mortality rate is because of the asexual erythrocytic stage of the life cycle of P. falciparum during which the parasite multiplies rapidly, increasing the number of merozoites in the human host. In the erythrocytic stage, merozoites periodically invade red blood cells (RBCs) every 44 to 48 hours, and this invasion lasts around one minute. Upon rupture of infected RBCs, merozoites become exposed to the host immune system for a brief interval (1–2 min) prior to invading new RBCs.3 The process is highly efficient and well-regulated, involving multiple crucial steps in a short period of time. P. falciparum whole genome sequencing4 and proteomic studies5 have revealed numerous essential genes and encoded proteins that play crucial roles in the invasion process. The invasion-related proteins are stored abundantly in specialized secretory apical organelles known as micronemes and rhoptries.6 Rhoptries, being the largest among the apical organelles, host multiple proteins. The rhoptry neck hosts and secretes mainly reticulocyte-binding homolog (Rh) family proteins7 [Rh1,8 Rh2a,9 Rh2b,9 Rh4,10 Rh511,12] and RON family proteins [RON2,13,14 RON4,15 RON5,16 RON6,17 RON11,18 RON1219] that play crucial roles in protein–protein interactions during the invasion process, and hence are considered as promising blood stage vaccine candidates. The structural and functional studies of these proteins have highlighted their unique role in the invasion mechanism.20–23 One such protein, P. falciparum rhoptry-associated leucine zipper-like protein 1 (PfRALP1), was previously identified as a rhoptry neck protein that plays a crucial role during invasion.24,25 However, the limited understanding of the structure and function of PfRALP1 warrants further investigation.

PfRALP1 is expressed in the late blood stage, mostly >40 h post-invasion, stored in the rhoptry neck region, and subsequently secreted to the merozoite surface during invasion.24PfRALP1 functions at the tight junction formation step by interacting with an unknown receptor protein present on the RBC membrane. Previous studies showed that antibodies raised against the C-terminal region of the PfRALP1 (PfRALP1-C1) significantly reduce merozoite invasion24 and exhibit strain-transcendent activity by targeting a conserved protein region.26 Additionally, semi-immune sera from malaria-infected asymptomatic populations are reactive towards PfRALP1-C1, suggesting that PfRALP1-C1 is immunogenic.24,25 Also, the PfRALP1 sequence is highly conserved across Plasmodium species and is refractory to gene deletion attempts.25 A recent study also demonstrated that glucosamine-induced PfRALP1 knockdown reduces invasion efficiency, parasite proliferation and schizont maturation.27 Thus, these unique features make PfRALP1 a potential vaccine candidate. However, thorough investigation of PfRALP1 is severely restricted as the pfralp1 gene contains 76.6% AT nucleotides,4,28 possesses low-complexity regions,29 and contains a disordered region,30 which makes it difficult to express in a heterologous system such as E. coli. The wheat germ cell-free extract system has emerged as an excellent expression system to synthesize difficult-to-obtain proteins.31,32 The PfRALP1-C1 protein has previously been expressed in the wheat germ cell-free extract system; however, quantitative data on expression yield was not reported, highlighting the need for alternative strategies for its production.25 Moreover, as compared to the bacterial expression system, the wheat germ cell-free extract system is known to be a low-yielding, expensive, and labor-intensive process.33 To overcome these challenges, herein we describe a viable semi-synthetic approach, by native chemical ligation (NCL)34 of two chemically synthesized peptide segments and a recombinant protein segment, to achieve high-yield synthesis of functional PfRALP1-C1.

NCL is a chemoselective reaction between an unprotected peptide α-thioester and an N-terminal cysteine-containing peptide under aqueous, near-neutral conditions, forming a native peptide bond. Owing to its mild reaction conditions and operational simplicity, NCL has been widely used for chemical protein synthesis. We synthesized PfRALP1-C1 by chemically conjugating appropriate α-thioester peptide segments with a recombinantly expressed protein segment by three-segment native chemical ligation.35–39 To enable NCL, we introduced non-native cysteine residues at each ligation junction: Asn439 was replaced with cysteine at the N-terminus of one of the chemically synthesized peptides (Seg 2), and Ser484 was replaced with cysteine at the N-terminus of the expressed protein segment (Seg 3). After ligation, we alkylated the cysteine residues to mimic native side chains. We report, for the first time, a comprehensive biophysical characterization of this semi-synthetic PfRALP1-C1. This study also includes functional characterization of the semi-synthetic construct using parasite growth inhibition assays. We showed that the semi-synthetic PfRALP1-C1 competitively inhibits the growth of the parasite in vitro.

Results and discussion

Bioinformatic analysis of PfRALP1

Primary sequence analysis revealed that PfRALP1 is a 749 amino acid-long protein that contains two carboxylate-rich negatively charged regions from residues 92 to 128 (glutamic acid-rich region) and residues 411 to 505 (an aspartic acid-rich region). The protein also harbors a leucine-rich region from residues 498 to 604, containing a highly conserved leucine-zipper domain from residues 511 to 532 and a coiled-coil domain from residues 595 to 624.24,25 To predict the secondary structure elements in full-length PfRALP1 (Fl-PfRALP1; residues 1 to 749) as well as the C-terminal region, PfRALP1-C1 (residues 396 to 637), we used bioinformatic tools such as PSIPRED,40 RaptorX41 and D2P2.42 The predicted secondary structure of Fl-PfRALP1 was predominantly contributed by intrinsically disordered regions and alpha helices (Fig. 1A and Fig. S1.1). Structure prediction of PfRALP1-C1 by ColabFold, which uses AlphaFold2 with MMseqs2,43–45 revealed the presence of both disordered and ordered regions, consistent with the results obtained using PSIPRED and RaptorX. The N-terminal region of the PfRALP1-C1 (residues 396 to 483) was completely disordered (Fig. 1B), likely due to low-complexity regions containing short tandem repeats of EDDHKN and GDDNKN, as well as a high frequency of charged amino acids (D, E, H, and K). PfRALP1-C1 retained an ordered region starting from residues 487 to 620, which possessed a leucine-zipper motif from residues 511 to 532. These ordered sequences formed a coiled-coil conformation contributed by a helix-loop-helix motif, where heptad repeats of leucine from residues 511 to 532 formed a leucine-zipper domain. Analysis of the ColabFold-AlphaFold2 predicted structure using ProteinTools46 revealed that the leucine heptad in the leucine-zipper domain contributed to non-polar interactions with the hydrophobic residues, mainly Leu or Ile, of the neighboring helix of the helix-loop-helix motif. Subsequently, hydrophobic clusters, comprising the side chains of Ile, Leu and Val, guided the formation of a stable core between the two helices in the folded state (Fig. 1C). Also, the presence of four clusters of salt-bridges, between residues Lys491, Asp519, and Glu522; between Asp495, and Lys515; between Lys545, and Glu548 and between Glu615, and Lys619, likely contributed to the overall stability of the protein conformation (Fig. 1D). In addition to the salt-bridge interactions mentioned above, H-bonding networks involving Gln531 and Asn535; Gln533 and Asn537; and Asn581 and Gln585 were evident (Fig. 1E).

Fig. 1. Bioinformatic analysis of PfRALP1. (A) Secondary structure prediction of the full-length PfRALP1 using PSIPRED and RaptorX. Highlighted portions (in grey and light orange colour) indicate the PfRALP1-C1 region. The N-terminal region in the PfRALP1-C1, predicted to be disordered, is shown in grey. The ordered regions are highlighted in light orange. Dashed lines: disordered region; H: helix; E: beta strand or extended region. (B) ColabFold-AlphaFold2 predicted the three-dimensional structure of PfRALP1-C1 with a pLDDT score showing model confidence. (C) Location of leucine heptads in the leucine-zipper motif (Leu511, Leu518, Leu525, Leu532; in red spheres) and the non-polar hydrophobic interactions established with the nearby hydrophobic residues (in green spheres). (D) Predicted four clusters for salt-bridge interactions and (E) additional hydrogen-bond networks were identified by using ProteinTools software.

Fig. 1

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Challenges in the recombinant expression of PfRALP1-C1

Prior to the semi-synthesis of PfRALP1-C1, we attempted to express this construct in E. coli, but this proved challenging, likely due to the presence of an 88-residue N-terminal disordered region. This region may serve as an excellent substrate for proteolytic cleavage or degradation, and may be toxic to the cell.47–49 Our initial approach for the recombinant expression of PfRALP1-C1 in E. coli involved incorporating either a His6 affinity tag or a His6-MBP affinity and solubility tag at the N-terminus. Although no overexpression was observed with the former tag (Fig. S2.1), the latter tag led to overexpression of PfRALP1-C1. However, purification was challenging as the expressed PfRALP1-C1 coeluted with the His6-MBP tag after treatment with tobacco etch virus (TEV) protease (Fig. S2.4–S2.7). Cell-free expression systems have been reported to be effective for obtaining such proteins.50,51 However, as mentioned earlier, a cell-free expression system is expensive, low-yielding and labor-intensive33 warranting an alternative approach for the production of PfRALP1-C1.

Semi-synthesis of PfRALP1-C1

We explored a new chemistry-driven semi-synthetic approach to obtain PfRALP1-C1. The synthetic strategy is shown in Scheme 1. The entire PfRALP1-C1 was assembled from three polypeptide segments using two NCL34 strategies combining two chemically synthesized segments with one expressed segment.52 The disordered region of PfRALP1-C1 (residues 396 to 483) was divided and synthesized into two peptide segments (Seg 1: residues 396–438, and Seg 2: residues 439–483), and the third polypeptide segment (Seg 3; residues 484 to 637) was expressed recombinantly in E. coli. Our design contains non-native N439C and S484C as temporary substitutions to carry out NCL, followed by alkylation of both cysteines to form pseudo-homoglutamine (Ψ-Gln).53,54 To efficiently generate peptide thioesters, peptide segments 1 and 2 were synthesized by Fmoc-based solid phase peptide synthesis (SPPS) using Fmoc-Dbz(Boc)-OH, a thioester surrogate known to be compatible with peptide synthesis at elevated temperatures on a Rink Amide aminomethyl resin.36,55 Details of the syntheses and HPLC purification are mentioned in the SI, Sections S3.4 and S3.5.

Scheme 1. Semisynthetic strategy for the convergent synthesis of PfRALP1-C1. (A) Architecture of the full-length PfRALP1 (Fl-PfRALP1), highlighting the C-terminal domain PfRALP1-C1. (B) Semisynthetic strategy to obtain PfRALP1-C1. The predicted disordered region (residue 396 to 483) was chemically synthesized into two polypeptide segments (Seg 1 and Seg 2). Seg 3 was recombinantly expressed in E. coli with a hexa-histidine (His6) tag followed by the TEV cleavage site at the N-terminus. TEV protease cleavage introduces a cysteine residue at the N-terminus of the expressed polypeptide to enable NCL. The cartoon diagram of the protein was generated using ColabFold-AlphaFold2. The red arrow is the cleavage site of the TEV protease. Detailed sequences for chemically synthesized Seg 1 and Seg 2, and for recombinantly expressed Seg 3 in E. coli, have been provided on the right-hand side.

Scheme 1

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The NCL of thioester peptide Seg 1-COSR (1′) and the N-terminal cysteine-containing peptide Seg 2-Dbz (2) in ligation buffer (6 M guanidine hydrochloride (Gu·HCl), 0.2 M phosphate buffer (PB), 5 mM 4-mercaptophenylacetic acid (MPAA), 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), pH 6.6) yielded product 3 (Seg 1-2-Dbz) within 42 hours (Fig. 2A(v)). The exogenous aryl thiol catalyst MPAA used for the NCL reaction was kept to a minimum to avoid interference during the subsequent one-pot Dbz activation step required for thioester generation. The ligation pH was also kept slightly below the standard pH 7.0, near the pKa of MPAA (∼6.6), as a compromise between ligation rate and competing thioester hydrolysis. Operating close to this pKa limits the concentration of both the reactive MPAA thiolate and hydroxide ion, thereby reducing the rate of hydrolysis alongside the rate of productive ligation. As a consequence, the ligation reaction was slow. We also observed partial hydrolysis of the thioester peptide 1′ into 1″, leaving unreacted cysteine-containing peptide 2 (Seg 2) in the reaction mixture. To generate the thioester at the C-terminus of the ligation product 3, the Dbz group was activated with NaNO2 in one-pot at pH 3, followed by treatment with sodium 2-mercaptoethane sulfonate (MESNa; pH 6.5) to obtain peptide 3′ (Seg 1-2-COSR) (Fig. 2A(vi)). The product 3′ was then purified by reverse-phase HPLC and 24.5 mg of the pure peptide 3′ (yield: 32.8% based on the limiting peptide 2) was isolated (Fig. 2A(vii)).

Fig. 2. Semi-synthesis of PfRALP1-C1. (A) Analytical HPLC chromatogram (wavelength = 214 nm) of (i) α-thioester Seg 1-COSR peptide (1′) and (ii) Seg 2-Dbz (2), which has an N-terminal cysteine. (iii) Within 10 min, the ligated product peak 3 started to appear. (iv) Within 6 h, a significant product peak 3 was observed in the ligation reaction medium containing ligation buffer (6 M Gu·HCl, 0.2 M PB, 5 mM MPAA, 5 mM TCEP, pH 6.6). (v) The ligation reaction was completed in 42 h. Some amount of peptide 2 remained unreacted in the mixture as the α-thioester peptide hydrolyzed to form 1″, which did not take part in the ligation reaction. (vi) One-pot thioester exchange of ligated product 3 was carried out in reaction buffer (6 M Gu·HCl, 0.2 M PB, 0.15 M MESNa, 0.07 M NaNO2, pH 5.2) to get peptide 3′, along with the thioester exchange of unreacted peptide 2 into 2′. (vii) HPLC-purified peptide 3′ along with ESI-MS data (calculated mass: 10133.05 Da, observed mass: 10132.92 ± 0.13 Da). (B) Analytical HPLC chromatogram (wavelength = 214 nm) of (i) purified peptide 3′ and (ii) recombinantly expressed and purified polypeptide Seg 3 (4). (iii) Chromatogram at the start (t = 0 min) of the second ligation with peptide 3′ and polypeptide 4 in ligation buffer (6 M Gu·HCl, 0.2 M PB, 50 mM MPAA, 10 mM TCEP, pH 6.8). (iv) 5 h after initiating ligation of 3′ and 4: major product 5, along with proposed lactam byproduct 3″ (from 3′) and residual unreacted 3* (MPAA-exchanged peptide 3′). (v) Ligation was completed in 20 h to form the final product 5 with unreacted polypeptide 4 due to the inaccessibility of hydrolyzed peptide 3″ for the ligation reaction. (vi) Product 5 was further purified with HPLC, and an alkylation reaction was done in reaction buffer (6 M Gu·HCl, 0.2 M PB, 2 equivalents of BrCH2CONH2, 1.1 equivalents of TCEP, pH 8). (vii) HPLC-purified product 6 with ESI-MS data. (Calculated mass: 27500.26 Da, observed mass: 27501.67 ± 1.04 Da). [‘*’ indicates MPAA, ‘**’ indicates MESNa byproduct].

Fig. 2

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To obtain recombinantly expressed polypeptide Seg 3 (residue 484 to 637) with an N-terminal cysteine for NCL, the polypeptide overexpression was carried out in Rosetta 2 (DE3) cells with an N-terminal hexa-histidine (His6) tag (for the purification) followed by a TEV protease cleavage site and a cysteine. After initial affinity chromatography, the expressed protein was treated with highly specific TEV protease, generating an N-terminal free cysteine in Seg 3 (Fig. S3.7.2). The tag-cleaved protein was further purified by (RP)-HPLC to obtain purified polypeptide 4 (Seg 3).

Chemoselective ligation of peptide 3′ (Seg 1-2-COSR) with Seg 3 (4), bearing an N-terminal cysteine, proceeded smoothly in the presence of 50 mM MPAA and 10 mM TCEP (Fig. 2B). Within 5 h, significant conversion with a new peak at 20.5 min corresponding to the final ligated product (5, Seg 1-2-3) with N439C and S484C substitution was observed (Fig. 2B(iv)). Prolonged reaction (20 h) led to the formation of a new peak (3″) with an observed mass of 9991 Da, consistent with loss of the MESNa leaving group (calc. 142.19 Da vs. observed mass difference of 142.05 Da), without concomitant addition of Seg 3, indicating consumption of peptide 3′via a pathway other than productive ligation. This mass corresponds to intramolecular cyclization of the thioester by the ε-amine of a proximal Lys residue to form a lactam, which would preclude further reaction with Seg 3 (Fig. 2B(v)). Upon completion, the final ligated product 5 was purified by (RP)-HPLC (Fig. 2B(vi)). Subsequently, the thiol groups of the non-native cysteines in 5 were alkylated with 2-bromoacetamide to avoid undesired side reactions from cysteine at physiological pH. The alkylated product 6 contained N439ΨQ and S484ΨQ, where cysteine residues were converted to pseudo-homoglutamine (ΨQ).53,54 The alkylated protein 6 was obtained in 71.4% yield (3.8 mg) after (RP)-HPLC purification and was characterized by ESI-MS (Fig. 2B(vii); calculated mass: 27500.26 Da, observed mass: 27501.67± 1.04 Da).

Given the high number of Asn-Gly and Asp-Gly motifs present in our sequence, aspartimide or associated byproduct formation was a concern during the ligation reactions. The formation of aspartimide and related byproducts has been shown to be minimized by restricting ligation temperature and reaction duration.56,57 Consistent with this, both ligations in this study were performed at room temperature for defined durations (42 h and 20 h, respectively). Definitive structural confirmation of aspartimide or iso-Asp-derived byproducts would require Asp-specific enzymatic assays or NMR analysis, which are beyond the scope of the present study. Therefore, the structural and functional integrity of the purified product 6 was instead assessed by circular dichroism and parasite growth inhibition assays, both of which support a correctly folded, structurally intact protein as detailed below.

Biophysical and functional characterization of the semi-synthetic PfRALP1-C1

To verify the predicted secondary structural fold, we measured the far-UV circular dichroism (CD) of the semi-synthetic PfRALP1-C1 (6) protein.58,59 Analysis of the CD spectrum revealed that it was predominantly α-helical with disordered regions, consistent with bioinformatic predictions. The profile showed two minima at around 219 nm and at 206 nm, suggesting the presence of mixed secondary structural elements (Fig. 3A). We deployed the web server BeStSel to estimate the secondary structure elements present in the semi-synthetic protein.60 The computed secondary structure from the CD raw data were 29% helix, 12% antiparallel β-sheet, 3% parallel β-sheet, 17% turn and 39% others (Fig. 3B). Also, from the thermal denaturation study using CD, we observed that the semi-synthetic PfRALP1-C1 displayed remarkable thermal stability and retained its secondary structure even at 90 °C (Fig. 3C).

Fig. 3. Characterization of the semi-synthetic PfRALP1-C1. (A) Far-UV CD spectra show predominant α-helical conformation. (B) Far-UV CD spectra, analyzed by the BeStSel web server, indicate the presence of α-helix, β-sheet, turns, and other secondary structures. (C) The thermal denaturation study shows that this protein is stable towards high temperatures up to 90 °C. (D) The growth inhibition study shows dose-dependent inhibition of parasite invasion into RBCs. PfRON22021–2059 was used as a positive control in this experiment. Experiments were performed twice independently, with triplicates for data reproducibility.

Fig. 3

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To test whether semi-synthetic PfRALP1-C1 could block merozoite invasion into RBCs, we assessed its activity in a parasite growth inhibition assay for one cycle with multiple concentrations in growth media. PfRON22021–2059 peptide (39 residues) was used as a positive control (Fig. 3D). We observed a dose-dependent inhibitory effect on parasite growth in the culture containing semi-synthetic PfRALP1-C1.

Conclusions

Earlier and recent studies have established that PfRALP1 is a novel tight-junction protein expressed during the schizont stage and localizes to the rhoptry neck until the merozoite engages in invasion, where it plays a key role. The invasion-related function of PfRALP1 is mediated by its C-terminal region (PfRALP1-C1), which contains an erythrocyte-binding epitope and predicted leucine-zipper/coiled-coil domains, although their relationship remains unclear. This region is also immunogenic and is therefore considered a potential blood-stage vaccine candidate against P. falciparum.

Overexpression of PfRALP1-C1 in E. coli proved challenging, possibly due to the presence of intrinsically disordered regions, as revealed by bioinformatic analysis and ColabFold-AlphaFold2 structural prediction, highlighting a limitation of the expression system. Our semi-synthetic approach was able to overcome this challenge. This approach highlights the utility of combining chemical synthesis and recombinant expression to obtain difficult-to-express malaria parasite proteins in good yield and purity. To the best of our knowledge, this is the first example of a malaria parasite protein obtained using a semi-synthetic approach. Given the limitations of conventional expression systems for such proteins, semi-synthesis provides a valuable alternative.

Structural characterization by CD indicates that the semi-synthetic PfRALP1-C1 predominantly adopts α-helix and disordered regions, consistent with the prediction by ColabFold-AlphaFold2. Functionally, the semi-synthetic protein competitively inhibits merozoite invasion into RBCs, suggesting that it retains binding epitope(s) involved in interactions with an as-yet unidentified receptor on RBCs.

Our study lays the groundwork for further research on the PfRALP1 protein aimed at defining the minimal region that is important for erythrocyte binding, identifying critical residues that directly participate in binding with the receptor, and, most importantly, elucidating the unknown receptor present on the erythrocyte and/or merozoite surface to which the PfRALP1 gets anchored. Together, our current study serves as the basis for advancing the molecular understanding of PfRALP1 function.

Experimental methods

General method for machine-assisted peptide synthesis

See SI Section S3 for detailed experimental procedures.

Circular dichroism and thermal denaturation

Far-UV CD spectra were recorded on a JASCO J-1500 spectropolarimeter equipped with a Peltier temperature controller. Lyophilized semi-synthetic PfRALP1-C1 was reconstituted in 10 mM phosphate buffer (pH 7.4) for a final concentration of 7.2 µM (0.2 mg ml−1) at 4 °C, as measured by UV absorption at 280 nm using a NanoPhotometer® NP80 (IMPLEN). The sample solution was then centrifuged at 13000 rpm at 4 °C for 10 min, and the spectra were recorded at room temperature (25 °C) in a quartz cuvette (1 mm path length) over 180–260 nm at 1 nm intervals with an average of three scans. The mean residue molar ellipticity was calculated using the following equation:Mean residue molar ellipticity = [ellipticity (mdeg) × Mw]/(10 × l × c × n)where ellipticity is the raw CD data recorded in millidegrees, Mw is the molecular weight of the protein, l is the path length (in cm) of the cuvette used, c is the concentration of protein in mg ml−1, and n is the number of residues in the protein. The secondary structure content was analysed using the BeStSel web server.

Growth inhibition assay (GIA)

Plasmodium falciparum 3D7 parasites were grown in complete RPMI 1640 media supplemented with 0.5% Albumax II and 0.005% hypoxanthine in RBCs with 2% hematocrit at 37 °C, as described earlier.55,61 In brief, a tightly synchronized early trophozoite-stage parasite culture was adjusted to 0.4% parasitemia and 2% hematocrit. For GIA, the culture was treated with semi-synthetic PfRALP1-C1 at 40 µM and 20 µM, with the protein dissolved directly in complete media. The intra-erythrocytic progression of the culture was monitored until the completion of one invasion cycle and the subsequent appearance of visible rings in the following cycle. In this experiment, a separate culture was maintained without protein treatment to serve as a control. After one invasion cycle was completed and visible rings were observed, small volumes from both treated and untreated cultures were collected to prepare smears on glass slides. Dry smears were then fixed with methanol and stained using Giemsa solution. Following washing and drying, the stained slides were examined under a light microscope to capture images for parasitemia calculation. The parasitemia was determined by counting ∼4000 erythrocytes using the Python script,62 with manual verification of the processed image output. Experiments were performed twice independently, with triplicate samples to ensure data reproducibility.

Author contributions

Conceptualization: A. N. & K. M.; investigation: A. N. & S. R.; formal analysis: A. N., S. R. & K. M.; project administration: S. R.; funding acquisition: K. M.; supervision: M. K. B. & K. M.; writing – original draft: A. N. with input received from all co-authors; writing – review & editing: A. N., S. R., & K. M.

Conflicts of interest

The authors declare no conflicts of interest.

Supplementary Material

CB-OLF-D6CB00176A-s001

Acknowledgments

We acknowledge the support from the Department of Atomic Energy, Government of India, under Project Identification No. RTI 40007. We extend our gratitude to the biophysical instrument facility at the Tata Institute of Fundamental Research Hyderabad.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). The SI contains bioinformatic analysis details, recombinant expression/purification data, synthetic procedures, HPLC/ESI-MS characterization, and biophysical/functional assay methods for semi-synthetic PfRALP1-C1. See DOI: https://doi.org/10.1039/d6cb00176a.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

CB-OLF-D6CB00176A-s001
CB-OLF-D6CB00176A-s001.pdf (2.3MB, pdf)

Data Availability Statement

The data supporting this article have been included as part of the supplementary information (SI). The SI contains bioinformatic analysis details, recombinant expression/purification data, synthetic procedures, HPLC/ESI-MS characterization, and biophysical/functional assay methods for semi-synthetic PfRALP1-C1. See DOI: https://doi.org/10.1039/d6cb00176a.

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