Bhattacharjya S, Awasthi S K, Adiga P R, Balaram P
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Protein Sci. 1998 Jan;7(1):123-31. doi: 10.1002/pro.5560070113.
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 CalphaH 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.
核黄素载体蛋白（RCP）在维生素B2跨胎盘膜运输过程中发挥着重要作用，这一过程对维持妊娠至关重要。维生素与载体蛋白的结合确保了最佳生物利用度，促进了运输。先前已将来自RCP的包含残基4 - 23（N21）、170 - 186（R18）和200 - 219（Y21）的三个抗原肽片段作为基于合成肽的避孕疫苗的潜在先导物进行了研究，本文使用圆二色光谱（CD）和核磁共振光谱（NMR）在水溶液以及结构稳定共溶剂三水合六氟丙酮（HFA）存在的情况下，对这三个片段的构象进行了研究。在pH 3.0的水溶液中，所有三个肽在很大程度上都是无结构的，R18和Y21肽的螺旋结构比例有限。通过CD实验估计，这两个肽的螺旋度百分比均为10%。加入HFA后，肽从无结构状态转变为螺旋状态，这一显著的结构转变通过Y21和R18在222 nm和208 nm处CD谱带的增强得以证明。在50% HFA（v/v）时结构转变完成，R18和Y21的螺旋度分别为40%和35%。即使在存在HFA的情况下，肽N21也没有明显的结构变化。在50% HFA中对这三个肽进行的NMR分析证实了R18和Y21的螺旋构象，这从CαH共振的高场位移以及许多连续的NH/NH核Overhauser效应（NOE）和许多中等距离NOE的存在中可以明显看出。对于这两个肽，螺旋构象在序列中心处已充分确立，而在两端都有明显的松散。NMR研究表明N21肽具有伸展构象。这两个肽（R18、Y21）的螺旋区域包含各自单克隆抗体识别的核心表位序列。这些结果为抗原肽的结构和折叠问题提供了一些启示。
