Alpha Helix Shortening in 1522 MSP-1 Conserved Peptide Analogs is Associated with Immunogenicity and Protection Against P. falciparum Malaria Marcia Cubillos, 1 Fabiola Espejo, 1 Jindra Purmova, 1 Juan C. Martinez, 2 and M.E. Patarroyo 1,2 * 1 Fundacio ´ n Instituto de Inmunologı ´a de Colombia (FIDIC), Bogota ´ , Colombia 2 Universidad Nacional de Colombia, Bogota ´ , Colombia ABSTRACT 1522 is a nonimmunogenic con- served high-activity binding peptide (HABP) belong- ing to Plasmodium falciparum MSP-1 protein N- terminal fragment. The key amino acids in binding to red blood cells (RBC) were identified and re- placed by others having similar mass but different charge. Because conserved HABPs are not antigenic nor immunogenic, immunogenicity and protectivity studies were then conducted on them in the Aotus monkey. 1 H-NMR studies included the lead peptide 1522 as well as the analogs 9782, 13446, 13448, and 13442 to relate their structure to biological function. All the peptides presented -helical structure, with differences observed in helix location and exten- sion. The nonprotective 1522 peptide was totally helical from the N- to the C-terminus, very similar to nonprotective 13442 and 13448 peptides whose exten- sion was almost totally helical. The 9782 and 13446 protective peptides, however, possessed a shorter helical region where modified critical binding resi- dues were not included. A more flexible region was generated at the C-terminus in those peptides with a shorter helical region, leading to a greater number of conformers. These data suggest that peptide flex- ibility results in increased interaction with immune system molecules, generating protective immunity. Proteins 2003;50:400 – 409. © 2003 Wiley-Liss, Inc. Key words: conformation; structure; receptor–li- gand; NMR; vaccine INTRODUCTION The identification of P. falciparum merozoite proteins binding to red blood cells (RBC) is part of our receptor– ligand interaction blocking strategy aimed at improving future antimalarial vaccine design. This disease affects 300 million people in the world, killing 2.5 million annually. 1 The proteins studied included merozoite sur- face protein-1 (MSP-1), 2 erythrocyte-binding antigen (EBA- 175 kDa), 3 glycophorin-binding protein (GBP-130), 4 mero- zoite surface protein-2 (MSP-2), 5 apical membrane antigen (AMA), 6 acid-basic repeat antigen (ABRA), 7 serine repeat antigen (SERA), 8 ring-infected erythrocyte surface anti- gen (RESA), 9 and some others. 10 MSP-1 is a 195-kDa merozoite membrane surface poly- morphic protein, having variable, semiconserved and con- served sequences 11 ; it suffers a series of proteolytic cleav- ages during its maturation. The first cleaves the protein into four fragments: the 83-kDa N-terminus, two central 30-kDa and 38-kDa fragments, and the 42-kDa C- terminus. 12 Immediately before invasion, the 42-kDa frag- ment is submitted to another cleavage resulting in 33-kDa and 19-kDa fragments. 13 Seventy-eight peptides (20 amino acids each) were syn- thesized according to the K 1 strain sequence to determine which of this protein’s amino acid sequences interact with RBC membrane in a specific receptor–ligand interaction. 2 All peptides were radiolabeled and analyzed in a specific binding assay developed for this purpose. One of these high-activity binding peptide (HABPs) was conserved pep- tide 1522 ( 202 QIPYNLKIRANELDVLKKLV 221 ) belonging to the 83-kDa N-terminal fragment, having a 150  13 nm (kDa) affinity constant capable of blocking in vitro parasite invasion of RBC by 60  14% at 200 M concentration. 2 Previous studies with this (and other peptides) have shown that conserved HABPs are not immunogenic. 14 Peptides analogous to 1522 were synthesized in an at- tempt to identify peptides that are both immunogenic and protective. Each one of peptide 1522 amino acids was replaced by glycine, and the resulting peptides were assayed for their ability to bind erythrocytes. In this study (and the previous study 14 ), such peptides modified with glycine were used in immunogenicity studies in mice and monkeys, allowing us to identify amino acids in peptide 1522 important for immunogenicity. These amino acids were replaced later by amino acids having similar mass but different charge and used in immunological studies for subsequent definitions of immunogenicity and protectivity characteristics. In the Aotus monkey experimental model, 1 H-NMR was then used to investigate the effect of these substitutions on peptide conformation. M. Cubillos and F. Espejo contributed equally to this work. *Correspondence to: Manuel Elkin Patarroyo, Fundacio ´n Instituto de Inmunologı ´a de Colombia (FIDIC), Cra 50 No. 26-00, Bogota, Colombia. E-mail: mepatarr@mail.com Received 20 May 2002; Accepted 1 October 2002 PROTEINS: Structure, Function, and Genetics 50:400 – 409 (2003) © 2003 WILEY-LISS, INC.