Sonication induced sheet formation at the air–water interface† K. S. Satheeshkumar and R. Jayakumar* Bio Organic Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India Received (in Cambridge, UK) 15th July 2002, Accepted 20th August 2002 First published as an Advance Article on the web 9th September 2002 A hydrophobic pentadecapeptide, AGAAAA- GAVVGGLGG (1), part of the prion sequence PrP (106-127), on fresh aqueous dissolution takes a mixture of random and sheet conformations which forms a stable monolayer with a high b-sheet content when compressed at the air–water interface. This also develops into a kinetically stabilized b-sheet structure on sonication. The secondary structure conversion of the prion protein in the normal form (PrP c ) into the abnormal form (PrP sc ) is reported to be a cause of several human and animal diseases, such as Creutzfeldt–Jacob disease, Gerstmann–Straussler–Scheinker syndrome, fatal familial insomnia, sheep scrapie, and bovine spongiform encephalopathy. 1 PrP c has high a-helix and low b- sheet content 2 in contrast to PrP sc , which is rich in b-sheets. 3,4 The reason for this conformational transition is still completely unknown despite numerous investigations. 5 In this work, we have shown that peptide 1 on ultrasonic treatment and compression at the air–water interface forms a stable b-sheet assembly. Insoluble or sparingly soluble polypeptides and proteins which form monolayers at the air–liquid interface are very applicable as models for biological systems to study structural transitions and orientation effects due to ‘molecular crowding’. 6–9 The sequence of the peptide used in this study is AGAAAA- GAVVGGLGG (1), which corresponds to H1 of human prion protein (residues 113-127). Peptide 1 was prepared by a solid phase method using Boc chemistry. The purity and composition were determined by amino acid analysis and were identified by MALDI-TOF MS analysis. Peptide 1 forms a stable film at the air–water interface. Fig. 1 shows a pressure–area isotherm for peptide 1. It is seen that the isotherm of peptide 1 is of the liquid expanded type (85 Å 2 ) indicating the fairly rigid nature of the film. The value of the molecular area of the monolayer can be directly evaluated from the intersection of the x-axis with the tangent line of the isotherm at the onset of the condensed phase of the film, and is found to be 30 Å 2 per molecule. The collapse pressure is in the region of 28 mN m 21 . The film formed by peptide 1 could be transferred to a quartz plate by y-type (vertical) deposition with a transfer ratio around 0.95. The circular dichroism (CD) spectrum of the transferred film (20 layers) is shown in the inset of Fig. 1. It is found that the peptide 1 solution spread at the air–water interface results in mono- layers containing predominant sheet conformation, as shown by the negative CD at 220 nm and the crossover at 213 nm. This indicates a profound effect of oriented molecular crowding on b-sheet assembly. The CD spectrum of peptide 1 in PBS at pH 7.4, (Fig. 2; curve a) exhibits a mixture of random, helix, sheet and turn conformations. The sheet content is ~ 30 %. Peptide 1 solution in vitro must be incubated for several days at ~ 37 °C and under basic conditions (pH 10.6) in order to build a significant b-sheet content. The CD spectrum of incubated protofibrils is given in the ESI.† On sonication it is known to produce energy rich vapour–liquid interfaces in the liquid with shear force (cavita- tion effect). We thought it would be worth investigating the effect of sonication on the conformation of peptide 1. To our surprise we found that sonication leads to kinetically stabilized b-sheet formation. The CD spectrum of peptide 1 in PBS at pH 7.4 is shown in Fig. 2 (curve a). The peptide solution was sonicated and the CD spectrum was recorded at 30 s intervals (Fig. 2, curves a to d). It is inferred from this study that upon sonication the sheet conformation predominates. This may be due to a highly energetic cavitational effect, which leads to interfacial effects on the aggregates. The plot of % sheet structure versus sonication time is shown in the inset of Fig. 2. It is tacit that the sheet structure of peptide 1 increases with sonication time. This † Electronic supplementary information (ESI) available: CD spectra and table of secondary structure motifs of peptide 1. See http://www.rsc.org/ suppdata/cc/b2/b206886a/ Fig. 1 Surface area–pressure isotherm for peptide 1. Inset: Circular dichroism (CD) spectrum of a 20 layer film of peptide 1. Fig. 2 CD spectra of peptide 1 in PBS at pH 7.4 and at different sonication time intervals: (a) 0 s, (b) 30 s, (c) 60 s, (d) 90 s. Inset: Plot of % sheet structure versus sonication time. % of secondary structure motifs of peptide 1 estimated by the SELCON program. 11 This journal is © The Royal Society of Chemistry 2002 2244 CHEM. COMMUN. , 2002, 2244–2245 DOI: 10.1039/b206886a