Fig 1.
(a) Flowchart of the peptide-optimization algorithm. (b) Structure of MBP in open (white, PBD code 1OMP) and closed (grey, PBD code 3MBP) configurations in which the coordinated maltotriose (red) and the Met330 (yellow) are highlighted.
Fig 2.
Computational generation of peptides.
Autodock Vina binding energy evolution as a function of the optimization steps for the generation of MBP-9. Arrows indicate the energy associated with the starting poly-alanine, MBP-9, and two peptides along the minimization path: MBP-10 and MBP-11.
Table 1.
Docking scores are calculated with Autodock Vina over 10 runs each generating 9 configurations, and with MC+Vina over the last 10 configurations of 10 runs. Errors are standard deviations over all the samples. In parentheses the protein configuration used for the docking. All the values are in kcal/mol.
Fig 3.
(a-b) Overlap of the 10 peptides final configurations obtained by redocking 10 times MBP-9 with the Vina+MC code for 100 steps at kBT = 0.6 on the Met330 site of the MBP (yellow) in open conformation (a) and in its maltotriose (red) containing closed conformation (b). (c) The MC+Vina calculated BE where the errorbars are the sums of the standard errors of the means calculated over the last 10 configurations of 10 runs of the docking protocols.
Table 2.
kD (SPR determined) and pI values for the MBP peptides.
CBE (Cannot be Estimated) is related to experiments in which signal variations were observed but KD estimation cannot be reached neither through kinetic nor saturation analysis.
Fig 4.
ESI-MS analysis of Protein-Peptide complexes.
Spectra of 20 μM MBP in 50 mM ammonium acetate pH7. (A) DP60 V, (B) DP 60 V in the presence of 20 μM MBP-9, (C) DP 180 V in the presence of 20 μM MBP-9, (D) DP 60 V in the presence of 20 μM MBP-9 and 20 μM maltose. In panel A the peaks are labeled by the charge acquired during the electrospray. In panels B-D the peaks corresponding to apoMBP (○), MBP:MBP-9 1:1 complex (•), MBP:MBP-9 1:2 complex (••), MBP:maltose 1:1 complex (gray solid circle) and MBP:MBP-9:maltose: 1:1:1 complex (black/gray solid circles) are labeled.
Fig 5.
Overlay of SPR sensorgrams for the interaction between the immobilized MBP protein and MBP-9 (A). The experimental curves corresponding to different concentrations of peptides (0–800μM) were fitted according to a single binding model with 1:1 stoichiometry. Competitive assay between peptides and maltose for immobilized MBP: Sensorgram of the co-injection of MBP-9 peptide at 200μM and maltose at 20mM (B).
Fig 6.
Tryptophan fluorescence quenching analysis showing the dose-response curve of the fluorescence values of MBP protein at 333nm plotted against the concentration values of MPB-9.
Fig 7.
Comparison between the experimental binding and the MC+Vina calculated BE.
The BE errorbars are standard errors of the means over 10 samples, while the experimental errobars are calculated as σΔG = 0.434(σkD/kD),. Docking into maltose site of peptides: (a) MBP-6, (b) MBP-1, (c) MBP-8, (d) MBP-3, (e) MBP-9. Highlighted with their Van der Waals spheres the MBP aromatic side chains involved in the binding (Tyr 155 219 34, Phe 337, Trp 62 340 230) and in black those capable of hydrogen bonding (Ser 337, Asp 14, Arg 66 344, Lys 297, Glu 153 44 45).