Single molecule force spectroscopy reveals the effect of BiP chaperone on protein folding

Abstract

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.

Mechanical properties of BiP protein determined by nano‐rheology

“…Recently, our group has been studying BiP binding properties, unfolding completely a protein substrate using single molecule optical tweezers. Our results showed that BiP binds to the unfolded state of MJ0366 substrate protein reversibly, preventing its refolding, and that this effect depends on both the type and concentration of nucleotides …”

“…Another interesting point to note is the allosteric communication between both BiP structural domains, which it has been described as an important feature that modulates BiP functionality. 3,6 In our group we have studied allosterism in both directions: first, from the NBD to the SBD, 15 studying BiP mechanical properties at the single molecule level with optical tweezer manipulation, and now in this work from the SBD to the NBD with ensemble measurements with nano-rheological experimental setup. This study has shown that there exists a communication between both domains, as when peptide is bound in the SBD, the affinity for ADP bound in the NBD is modified (evidenced by the drop in K D value), demonstrating that SBD is allosterically coupled to NBD, as described in previous studies.…”

“…Interesting and novel approaches to study dynamics in chaperones have arised combining spectroscopic techniques, like Heteronuclear Single Quantum Coherence Spectroscopy (HSQC) with computational simulations . A different and novel approach to study mechanical properties of proteins has been the manipulation of single molecules subjected to force application . Recently, our group has been studying BiP binding properties, unfolding completely a protein substrate using single molecule optical tweezers.…”

“…[12][13][14] A different and novel approach to study mechanical properties of proteins has been the manipulation of single molecules subjected to force application. 15 Recently, our group has been studying BiP binding properties, unfolding completely a protein substrate using single molecule optical tweezers. Our results showed that BiP binds to the unfolded state of MJ0366 substrate protein reversibly, preventing its refolding, and that this effect depends on both the type and concentration of nucleotides.…”

“…Our results showed that BiP binds to the unfolded state of MJ0366 substrate protein reversibly, preventing its refolding, and that this effect depends on both the type and concentration of nucleotides. 15 Taking all this in consideration, and assuming that BiP mechanical properties are beginning to be understood, our group has been trying to find new molecular descriptors about BiP acting as a chaperone and as a molecular motor during ER translocation. 16 To achieve this, in this study, we have focused on studying how BiP's mechanical properties are correlated with nucleotides and substrate binding, considering modifications in the internal viscosity of the folded state of BiP, and how these properties modulate its function.…”