Deciphering Molecular Interactions of Native Membrane Proteins by Single-Molecule Force Spectroscopy

Abstract

Molecular interactions are the basic language of biological processes. They establish the forces interacting between the building blocks of proteins and other macromolecules, thus determining their functional roles. Because molecular interactions trigger virtually every biological process, approaches to decipher their language are needed. Single-molecule force spectroscopy (SMFS) has been used to detect and characterize different types of molecular interactions that occur between and within native membrane proteins. The first experiments detected and localized molecular interactions that stabilized membrane proteins, including how these interactions were established during folding of alpha-helical secondary structure elements into the native protein and how they changed with oligomerization, temperature, and mutations. SMFS also enables investigators to detect and locate molecular interactions established during ligand and inhibitor binding. These exciting applications provide opportunities for studying the molecular forces of life. Further developments will elucidate the origins of molecular interactions encoded in their lifetimes, interaction ranges, interplay, and dynamics characteristic of biological systems.

Investigation of the Interaction between a Bivalent Aptamer and Thrombin by AFM

“…35,36 SMFS Reveals the Interaction Pattern of the ThrombinÀAptamer Bond. When the thrombin and Bi-8S aptamer were set in buffer solution without blocking aptamers (Figure 3a), two pronounced force peaks were clearly visible in the superimposition at the most probable contour lengths of 13 and 28 nm, which is in agreement with the expected contour length.…”

Point Mutations in Membrane Proteins Reshape Energy Landscape and Populate Different Unfolding Pathways

“…9 The forced unfolding of membrane proteins tethered to an AFM cantilever stylus is, however, directional, and occurs via sequential unfolding steps of well-defined structural segments composed of hydrophilic interhelical loops, transmembrane α-helices or α-helical pairs. Each such structural segment has a certain probability of unfolding individually or together with adjacent segments.…”

“…Since each unfolding step has a certain probability to be taken, this is also the case for the unfolding pathway. 9 Experiments on bacteriorhodopsin (BR), 10,11 NhaA 12 and bovine rhodopsin 13,14 have shown that small changes in the environment can alter interactions stabilizing the structural segments within membrane proteins. We have recently shown that inter-and intramolecular interactions contribute differently towards stabilizing these structural segments.…”

Cholesterol increases kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor

“…Intra-and intermolecular interactions stabilizing membrane proteins can be quantified and localized using atomic force microscopy (AFM) -based single-molecule force spectroscopy (SMFS) (31,32). SMFS enables the assignment of these interactions to individual secondary structure elements, such as transmembrane α-helices, β-strands, or polypeptide loops of membrane proteins embedded in lipid membranes.…”

“…1B). An unfolding step, in which a stable structural segment unfolds, describes the transfer of one unfolding intermediate to the next intermediate (31). To assign the unfolding steps and stable structural segments, every force peak of an F-D curve was fitted using the worm-like chain (WLC) model (Materials and Methods).…”