Fig 1.
A preview of the protein aggregation/fibrillation pathway and chemical structures of the small aroma producing molecules used in this study.
(A) A general overview of the steps involved in globular protein aggregation/fibrillation. The pathway of assembly varies in different references, details may change i.e. some references use protofibrils as synonym for oligomers [9], however the theory generally holds to be reliable. (B) 2D chemical structures of the two polyphenols and the polyamine used in this study.
Fig 2.
(1) 5 ml of HEWL at 2 mg/ml was initially added to the bottom of a 100 ml Duran bottle; (2 and 3) Small and large holes were made in 50 ml falcon tubes, respectively; (4 and 5) The falcon tubes with holes were placed into Duran bottles containing HEWL; (6) 50 μl volume of PEA, Cin or TEMED were added to the falcon tubes with small (S) and large (L) holes and placed inside the Duran bottle containing HEWL; (7–10) The lids of the falcon tubes and Duran bottles where sealed together and incubated at 54°C for 24 hours in a shaker at 150 rpm for the process of aggregation or fibril formation to take place and the effect of the aroma of PEA, Cin or TEMED in preventing fibrillation to be assessed.
Fig 3.
Inhibition of HEWL fibril formation.
(A) Effect of PEA, Cin and TEMED aroma on HEWL amyloid fibrillation detected by ThT emission. (B) Percentage of inhibition in the presence of either small or large amounts of aroma on HEWL fibril formation. HEWL samples were incubated for 24 hours at pH 2.2 and 54°C (except for the not-heated sample) and ThT emission measured at 484 nm.
Fig 4.
Analysis of hydrophobic patches and secondary structure of HEWL treated with aroma.
(A) Effect of PEA, Cin and TEMED aroma on the surface hydrophobicity of HEWL. Changes in Nile red emission spectra obtained in the presence of HEWL at pH 2.2 when not-heated or incubated at 54°C with or without different aroma. Further details are given in the ‘Experimental procedures’ section of this manuscript. (B) Far-UV CD spectra of HEWL incubated with or without aroma. Secondary structures of not-heated HEWL in comparison with incubated HEWL after 24 hours with or without aroma.
Fig 5.
Size distribution of HEWL as revealed by DLS.
The panels represent the DLS graphs of size distribution of HEWL particles for not-heated HEWL (A); not-treated HEWL incubated for 24 hours (B); HEWL treated with PEA-L aroma (C); HEWL treated with PEA-S aroma (D); HEWL treated with Cin-L aroma (E); HEWL treated with Cin-S aroma (F); HEWL treated with TEMED-L aroma (G); and HEWL treated with TEMED-S aroma (H). Measurements of HEWL sample solutions were done at 2 mg/ml.
Fig 6.
AFM analysis of HEWL incubated with or without aroma.
AFM images of not-heated HEWL (A); not-treated HEWL (B); HEWL treated with PEA-L aroma (C); HEWL treated with PEA-S aroma (D); HEWL treated with Cin-L aroma (E); HEWL treated with Cin-S aroma (F); HEWL treated with TEMED-L aroma (G); and HEWL treated with TEMED-S aroma (H).
Fig 7.
(A) Rate of lysis of Micrococcus lysodeikticus (M. luteus) as a function of HEWL, pH = 6.2, 25°C. (B) The relative activity (%) (With respect to the not-heated enzyme) of HEWL with or without varying aroma treatments. (C) Changes in the slope of activity plot as a representative of enzyme activity in different conditions.
Fig 8.
Effect of aroma of HEWL as assessed by SDS-PAGE.
(A) Gel lanes are as follows: Protein marker (M), not-heated HEWL (1), not-treated HEWL (2), HEWL treated with different aroma (lanes 3–8); PEA-L, PEA-S, Cin-L, Cin-S, TEMED-L and TEMED-S, respectively). (B) Colour of samples 1–8 with added loading dye for SDS-PAGE analysis.
Fig 9.
A possible mechanism of the effect of aroma from a phenol, an aldehyde and a diamine in preventing HEWL fibril formation. All pathways were concluded using results from several different experiments.