Monitoring the conformational changes of an intrinsically disordered peptide using a quartz crystal microbalance

Abstract

Intrinsically disordered peptides (IDPs) have recently garnered much interest because of their role in biological processes such as molecular recognition and their ability to undergo stimulus-responsive conformational changes. The block V repeat-in-toxin motif of the Bordetella pertussis adenylate cyclase is an example of an IDP that undergoes a transition from a disordered state to an ordered beta roll conformation in the presence of calcium ions. In solution, a C-terminal capping domain is necessary for this transition to occur. To further explore the conformational behavior and folding requirements of this IDP, we have cysteine modified three previously characterized constructs, allowing for attachment to the gold surface of a quartz crystal microbalance (QCM). We demonstrate that, while immobilized, the C-terminally capped peptide exhibits similar calcium-binding properties to what have been observed in solution. In addition, immobilization on the solid surface appears to enable calcium-responsiveness in the uncapped peptides, in contrast to the behavior observed in solution. This work demonstrates the power of QCM as a tool to study the conformational changes of IDPs immobilized on surfaces and has implications for a range of potential applications where IDPs may be engineered and used including protein purification, biosensors, and other bionanotechnology applications.

Block V RTX Domain of Adenylate Cyclase from Bordetella pertussis: A Conformationally Dynamic Scaffold for Protein Engineering Applications

“…The folding of the RTX domains could then be monitored by the change in the resonance frequency of the peptides as the β-roll formation affected the local mass and viscosity near the crystal surface. It was found that the quartz crystal could also serve as a capping group, stabilizing the C-terminus of the peptide and allowing for calcium-dependent folding ( Figure 2 A) [ 71 ]. Interestingly, some calcium-dependent changes were also seen when the peptides were immobilized by the N-terminus (and the C-terminus was uncapped) and it was assumed this was due to a molecular crowding effect on the surface.…”

“…Cyan fluorescence protein (CYP) and yellow fluorescence protein (YFP) were used to characterize the conformational change of the domain by FRET, and this led to the discovery that YFP could serve as a C-terminal capping group [ 70 ]. Tethering of the RTX domain to a QCM crystal allowed the surface to function as a terminal capping group [ 71 ]; ( B ) rearranged RTX domains were created to explore sequence modularity and functionality. Rearranged RTX repeats were built with or without the capping group [ 64 ]; and ( C ) different numbers of full-length RTX domains (each with C-terminal caps) were concatenated with linkers and used in biomolecular recognition and protein hydrogel studies [ 68 , 69 ].…”

Monitoring structural changes in intrinsically disordered proteins using QCM-D: application to the bacterial cell division protein ZipA

“…To date there has been a limited number of reports 23,24 regarding IDP conformation studies using acoustic biosensors. Here we extend the discrete-molecule approach to the real time study of IDPs reversible expansion and collapse.…”

Using a second‐order differential model to fit data without baselines in protein isothermal chemical denaturation

“…[17][18][19][20] Evaluation on protein unfolding/folding energy is one of primary methods to understand protein conformational stability, 21 protein mutation effects, 22 and property changes of small compounds binding to protein. 23,24 Protein unfolding/folding commonly follows a canonical two-state mechanism, 25 where protein cooperatively transits from folded state (F or native state) to unfolded state (U or denatured state) during unfolding in a changing environment (or vise versa in protein (re)folding).…”