Mechanical Properties of BiP (Binding Immunoglobin Protein)

Abstract: Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to

the Hsp70 family, involved in the regulation of important biological processes such as synthesis,

folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved

domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding

Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the

NBD, and BiP’s extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP

function depends on both structural and catalytic properties that may contribute to its performance.

One novel approach to study the mechanical properties of BiP considers exploring the

changes in the viscoelastic behavior upon ligand binding, using a technique called nano-rheology.

This technique is essentially a traditional rheology experiment, in which an oscillatory force is

directly applied to the protein under study, and the resulting average deformation is measured. Our

results show that the folded state of the protein behaves like a viscoelastic material, getting softer

when it binds nucleotides- ATP, ADP, and AMP-PNP-, but stiffer when binding HTFPAVL peptide

substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP,

decreasing it dissociation constant (KD) around 1000 times, demonstrating allosteric coupling

between SBD and NBD domains.