Single molecule force spectroscopy reveals the effect of BiP chaperone on protein folding.
BiP (Immunoglobulin Binding Protein) is a member of the Hsp70 chaperones that participates in protein folding in the endoplasmic reticulum. The function of BiP relies on cycles of ATP hydrolysis driving the binding and release of its substrate proteins. It still remains unknown how BiP affects the protein folding pathway and there has been no direct demonstration showing which folding state of the substrate protein is bound by BiP, as previous work has used only peptides. Here, we employ optical tweezers for single molecule force spectroscopy experiments to investigate how BiP affects the folding mechanism of a complete protein and how this effect depends on nucleotides. Using the protein MJ0366 as the substrate for BiP, we performed pulling and relaxing cycles at constant velocity to unfold and refold the substrate. In the absence of BiP, MJ0366 unfolded and refolded in every cycle. However, when BiP was added, the frequency of folding events of MJ0366 significantly decreased, and the loss of folding always occurred after a successful unfolding event. This process was dependent on ATP and ADP, since when either ATP was decreased or ADP was added, the duration of periods without folding events increased. Our results show that the affinity of BiP for the substrate protein increased in these conditions, which correlates with previous studies in bulk. Therefore, we conclude that BiP binds to the unfolded state of MJ0366 and prevents its refolding, and that this effect is dependent on both the type and concentration of nucleotides.
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, RM, Chile.|Laboratory of Cellular and Molecular Biophysics, School of Physics & Astronomy, University of Minnesota, Twin Cities, Minnesota.
María Paz
MP
Ramírez
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, RM, Chile.
Maira
M
Rivera
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, RM, Chile.
Diego
D
Quiroga-Roger
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, RM, Chile.
Andrés
A
Bustamante
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, RM, Chile.
Marcela
M
Vega
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, RM, Chile.
Mauricio
M
Baez
Laboratory of Cellular and Molecular Biophysics, School of Physics & Astronomy, University of Minnesota, Twin Cities, Minnesota.
Elias M
EM
Puchner
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, RM, Chile.
Christian A M
CAM
Wilson
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D063747:Methanocaldococcus / Q000737:chemistry* / Q000235:genetics; D008956:Models, Chemical; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins
BiP chaperone; binding parameters; force spectroscopy; nucleotides dependence; optical tweezers