Fig 1.
(A) Molecular Structure of BiP (Protein Data Bank ID: 5E84). (B) Domain architecture of BiP. Nucleotide binding domain (NBD) is shown in blue color, whereas the SBD-α and SBD-β are shown in green and purple colors, respectively. Hydrophobic linker connecting the NBD and SBD is shown in red color.
Fig 2.
Expression and purification of BiP.
(A) SDS-PAGE of purified BiP. Lane labeled M contained the molecular weight markers. (B) Mass spectrometry of intact full length BiP confirmed the purity of the protein.
Fig 3.
Structural characterization of the wild-type and chemically modified BiP proteins.
(A) SDS-PAGE of wild-type and chemically modified BiP with increasing levels of 4-ONE. Increasing 4-ONE shows increasing loss of monomer protein. (B) Far-UV circular dichroism (CD) spectra of wild-type (black) and chemically modified BiP (red). (C) Fluorescence spectra of wild-type BiP (black) and chemically modified BiP (red).
Fig 4.
(A) Changes in the CD signal at 222 nm as a function of urea concentration. (B) Changes in the CD signal at 222 nm as a function of solution temperature. (C) Changes in the protein fluorescence at 364 nm as a function of urea concentration. (D) Changes in the protein fluorescence at 330 nm as a function of solution temperature. (E) Changes in the specific heat capacity of the protein as a function of increasing temperature as measured by DSC. The deconvolution fits are indicated as dashed lines. In all panels, black and red colors indicate the data for wild-type BiP and chemically modified BiP, respectively.
Fig 5.
(A) Changes in the near-UV CD signal of BiP at 266nm as a function of varying ATP concentration. (B) Changes in ATPase activity of protein before and after chemical modification. Blue bar is ATP only. (C) Changes in fluorescence anisotropy of peptide HTFPAVL at 482nm as a function of varying BiP concentration. (D) Changes in light scattering at 350 nm due to the aggregation of citrate synthase as a function of incubation time at 43°C. Blue indicates citrate synthase alone. In all panels, black and red colors indicate the data for wild-type BiP and chemically modified BiP, respectively.
Fig 6.
(A) Crystal structure of BiP indicating all chemically modified residues (red) and residues in ATP binding pocket (yellow). (B) Interactions between Trp103 and chemically modified Lys123 in NBD. (C) Trp600 in SBD does not form any interactions with chemically modified lysines. (D) Nucleotide binding domain (NBD) showing stabilizing interactions between key residues in the ATP binding pocket (yellow) and chemically modified lysine residues (red). (E) Substrate binding domain (SBD-β) showing no interactions between the loops responsible for immunoglobulin binding (green) and chemically modified residues (red). (F) 5-residue average hydrophobicity plot (calculated using the program Accelrys Discovery Studio Visualizer) of BiP indicating the location of chemically modified residues (red).