Fig 1.
A) PCB chemical structure with labeled (A-D) pyrolle rings (oxygen atoms are in red and nitrogen in blue); B) Schematic of HSA structure with domains and ligand binding sites labeled; Radius of gyration (Rg) values (C) and 2D RMDS plots for free HSA (D), HSA-PCB(IIA) (E), and HSA-PCB(IB) (F) during 300 ns molecular dynamic simulation.
Fig 2.
Calculated RMSF values (Å) from molecular dynamics simulation of free HSA, HSA-PCB(IB), and HSA-PCB(IIA).
RMSF values along whole protein sequence (A), sum of RMSF values of each HSA domain (B), and difference of RMSF values between PCB-HSA complexes and free HSA, with marked amino acid (aa) residues involved in PCB binding for site IB (C), and for site IIA (D).
Fig 3.
Conformational changes of free HSA (A), HSA-PCB(IB) (B), and HSA-PCB(IIA) (C) during molecular dynamics simulation. The starting structures (0 ns) are represented in grey and the structures from the last snapshot (300 ns) in black (free HSA), blue (HSA-PCB(IB)) and red (HSA-PCB(IIA)). The red arrows direct to regions of PCB-bound HSA with increased flexibility, and blue one to decreased flexibility compared to flexibility of free HSA (black arrows); Conformational changes of PCB bound to site IB (D) and site IIA (E) during molecular dynamics simulation. The starting structures (0 ns) are represented in grey and the structures from the last snapshot (300 ns) in blue (PCB-IB) and red (PCB-IIA); (F) Absolute values of dihedral angles of starting PCB structure and of average dihedral angles calculated before and after PCB conformational change (at 107 ns for PCB-IB and at 169 ns for PCB-IIA).
Fig 4.
(A) UV-VIS spectra of free HSA, free PCB, and HSA-PCB complex (molar ratio 1:1); (B) Far-UV circular dichroism spectra of HSA in the absence (a) and presence (b) of PCB. Dot line curve shows spectrum of PCB; (C) The FT-IR spectra of free HSA (solid line), and the FT-IR difference spectra of HSA-PCB complex (molar ratio 1:1) (dot line). All spectra were recorded in 20 mM Tris buffer (pH 7.4) with samples of 18 μM HSA and 18 μM PCB.
Fig 5.
(A) Temperature dependence of 0.5 μM HSA ellipticity at 222 nm in the presence and absence of 0.5 μM PCB; (B) Temperature dependence of 1 μM HSA fluorescence at 340 nm in the presence and absence of 1 μM PCB (λEX = 280 nm); (C) SDS-PAGE profile after trypsin digestion of 3.8 μM HSA in the presence and absence of 3.8 μM PCB.
Lane K: HSA without trypsin; lane M: MW markers; lanes 1–6 correspond to digestion times 0.5, 2, 5, 10, 30, and 60 min without PCB, respectively; lanes 7–12 correspond to digestion times 0.5, 2, 5, 10, 30 and 60 min with PCB, respectively.
Table 1.
Binding scores (kcal mol-1) of different ligands docked to free and phycocyanobilin (PCB)-bound human serum albumin (HSA; at site IB or IIA), before and after 300 ns of molecular dynamics (MD) simulation.