Atomic Force Spectroscopy in Biological Complex Formation: Strategies and Perspectives

Abstract

Atomic force spectroscopy has become a widely used technique for investigating forces, energies, and dynamics of biomolecular interactions. These studies provide dissociation kinetic parameters by pulling apart proteins involved in a complex. Biological complexes are studied under near-physiological conditions, without labeling procedures, and are probed one at time, the latter allowing to one obtain results which are not averaged over the ensemble. However, to gain reliable information, some experimental aspects have to be carefully controlled. In particular, the immobilization of molecular partners to AFM tips and supports, required to force the molecular dissociation, plays a crucial role in determining the success of the experiments. To actually resolve single interactions, multiple simultaneous complex dissociations have to be avoided, and nonspecific adhesions, commonly found in these studies, have to be recognized and discarded. This article is aimed at offering a critical revisitation of the atomic force spectroscopy technique applied to the study of biomolecular interactions, highlighting the critical points, identifying strategies to be adopted for a more reliable data extraction and interpretation, and pointing out the experimental and theoretical aspects which still need to be refined. To this purpose, we take advantage of the vast landscape of literature and then proceed into the details of our works. In this respect, we describe the general principles of the technique, the procedures for protein immobilization, and how they can affect the results. We emphasize the use of computational docking to predict molecular complex configurations, when unknown, as a useful approach to select proper anchorage architectures. Additionally, we deal with data acquirement and analysis, with regard to the force curve selection, to the force histograms interpretation, and to the theoretical frameworks used to extract kinetic parameters. Through this, we outline that AFS can be successfully used both to investigate complexes having very different affinities and also to reveal competitive binding mechanisms, thus gaining deeper information about molecular interactions.

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy

“…25,32 In a typical AFS experiment, a ligand is anchored to the AFS tip while the receptor is immobilized onto a substrate. Force-distance curves are thus performed cyclically on the tethered system, as sketched in Figure 2 and described in the legend.…”

“…As collected force curves can significantly differ in AFS unbinding experiments in the single molecule regime, 25,32 force curves were accepted whose retraction portion, before the jump-off, exhibited a nonlinear trend starting and ending at the zero-deflection line where the nonlinear trend is attributed to the viscoelastic properties of the PEG linker under stretching. 36,37 When multiple jumps were observed, which could be due to subsequent rupture of the complex bonds, the curves were accepted if the last jump started and ended at zero deflection, with the last jump taken as representative of the unbinding process.…”

Binding ability of chitinase onto cellulose: an atomic force microscopy study

“…[6][7][8] In our previous work, 9 we applied force spectroscopy to measure the interaction force between chitin and two different kinds of chitin-binding domains (ChBDs) of a multidomain chitinase. The chitinase is produced by Thermococcus kodakarensis KOD1, a hyperthermophilic archaeon, and is composed of five characteristic domains, including dual catalytic domains (CatD A and B) and triple ChBDs (ChBD1, ChBD2 and ChBD3).…”

A physical picture for mechanical dissociation of biological complexes: from forces to free energies

“…2(B). We use a PEG fingerprint to select the curves containing specific rupture events [24][25][26] , which are characterized by a WLC-like increase in the force with the persistence and contour length of the polymer spacer (0.37nm and 20nm respectively) 43,44 . Furthermore, a careful orientation protocol was designed to increase the ratio of specific interactions, typically low.…”

“…In this work, we study the problem of mechanical dissociation of molecular complexes 10,[22][23][24][25][26] . Here, the force is typically exerted to the complex through a polymer linker attached to the Table 1 Free energy barrier height ∆G † , position x † and dissociation free energy ∆G 0 for some biomolecular complexes.…”