Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone

Abstract

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B₂ across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of C^αH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides.