Molecular Force Spectroscopy

Molecular interactions drive all processes in life. They determine the molecular crosstalk and build the basic language of biological processes. Molecular interactions fold the polypeptide into the functional protein, stabilize the structure, or lead to protein misfolding. These molecular forces determine protein-protein interactions, switching on and off ion channels, ligand-binding, the functional states of receptors, and the supramolecular assembly of molecular machines to functional units. On more complex scales molecular interactions guide cell adhesion, migration, communication and differentation. Other examples show how they sculpture reaction pathways of biological processes. Because of this enormous importance it is one pertinent demand in life sciences to characterize how these interactions are established and how they guide biological processes ranging from the molecular to cellular scale. To decipher fundamentals of this biological language at the molecular scale, we have pioneered atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) of native membrane proteins.

By applying SMFS to membrane proteins we characterize their unfolding and folding pathways into the lipid or native membrane at physiological relevant conditions (e.g., buffer solution, room temperature or 37°C). Alternatively we also apply SMFS to quantify and structurally localize inter- and intramolecular interactions of membrane proteins. The structural localization allows to map these interactions to single alpha-helices, beta-strands or polypeptide loops.

Single-molecule force spectroscopy (SMFS) for the manipulation, control and design of biological systems. (a) Measuring interaction forces F of single biomolecules by AFM. The examples shown probe ligand–receptor interactions in their isolated form (left) and embedded in the cellular environment (the AFM tip has been replaced by a biological cell); stretching of DNA; unfolding of Ig27-titin tandem constructs; and unfolding of a transmembrane protein (MP) embedded in the lipid or cellular membrane. (b) SMFS can detect and locate interactions (circles) on the teritiary structure of membrane proteins (here proton/sodium antiporter NhaA from E. coli). A ligand (sodium ion) or inhibitor (2-aminoperimidine, AP) binding to the ligand-binding site (large green circle) establishes different interactions activating (large green circle) or deactivating (large green and red circles) the antiporter. c, Single molecule cut and paste assay developed by the Group of Herman Gaub (LMU Munich) to mechanically pick up single molecules from discrete storage sites with a DNA oligomer functionalized to the AFM probe and depositing the molecules at a target site with nanoscopic precision. Figure adapted from Muller & Dufrene, Nature Nanotechnology (2008) 3, 261-269. — Single-molecule force spectroscopy (SMFS) for the manipulation, control and design of biological systems. (a) Measuring interaction forces F of single biomolecules by AFM. The examples shown probe ligand–receptor interactions in their isolated form (left) and embedded in the cellular environment (the AFM tip has been replaced by a biological cell); stretching of DNA; unfolding of Ig27-titin tandem constructs; and unfolding of a transmembrane protein (MP) embedded in the lipid or cellular membrane. (b) SMFS can detect and locate interactions (circles) on the teritiary structure of membrane proteins (here proton/sodium antiporter NhaA from *E. coli*). A ligand (sodium ion) or inhibitor (2-aminoperimidine, AP) binding to the ligand-binding site (large green circle) establishes different interactions activating (large green circle) or deactivating (large green and red circles) the antiporter. c, Single molecule cut and paste assay developed by the Group of Herman Gaub (LMU Munich) to mechanically pick up single molecules from discrete storage sites with a DNA oligomer functionalized to the AFM probe and depositing the molecules at a target site with nanoscopic precision. Figure adapted from Muller & Dufrene, Nature Nanotechnology (2008) 3, 261-269.

Molecular Force Spectroscopy

Further reading

YidC assists the stepwise and stochastic folding of membrane proteins

Molecular plasticity of the human voltage-dependent anion channel embedded into a membrane

Quantifying and identifying two ligand-binding sites while imaging native human membrane receptors by AFM

Impact of holdase chaperones Skp and SurA on the folding of β-barrel outer membrane proteins

Single-molecule force spectroscopy of membrane proteins from membranes freely spanning across nanoscopic pores

Dynamic single-molecule force spectroscopy of rhodopsin in native membranes

Observing a lipid-dependent alteration in single lactose permeases

Action of the Hsp70 chaperone system observed with single proteins

Substrate-induced changes in the structural properties of LacY

The peptide transporter DtpA populates two alternate conformations from which inhibitor binding promotes one

Mechanistic explanation of different unfolding behaviors observed for transmembrane and soluble β-barrel proteins

Single-molecule force spectroscopy of G-protein coupled receptors

Nanomechanical properties of proteins and membranes depend on loading rate and electrostatic interactions

Structural, kinetic, and mechanical differences between dark-state rhodopsin and opsin

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

Out but not in: β-strands shape the unfolding pathway but not the refolding of the large transmembrane β-barrel protein FhuA

Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase

Ligand-specific interactions modulate kinetic, energetic and mechanical properties of the human β2 adrenergic receptor

Structural, energetic and mechanical perturbations in a rhodopsin mutant that causes congenital stationary night blindness

Single-molecule force spectroscopy from nanodiscs – an assay to quantify folding, stability and interactions of native membrane proteins

KpOmpA a transmembrane protein anchoring the outer membrane of Klebsiella pneumoniae unfolds and refolds in response to tensile load

Locating an extracellular K⁺-dependent interaction site that modulates betaine-binding of the Na⁺-coupled betaine symporter BetP

Competing interactions stabilize pro- and anti-aggregant conformations of human Tau

One β-hairpin after the other: Exploring refolding pathways and kinetics of the transmembrane β-barrel protein OmpG

High-resolution atomic force microscopy and spectroscopy of native membrane proteins

Five challenges to bringing single-molecule force spectroscopy into the living cell

Conservation of molecular interactions stabilizing bovine and mouse rhodopsin

A ”force buffer” protecting immunoglobulin titin

Assessing the structure and function of single biomolecules with scanning transmission electron and atomic force microscopes

Dual energy landscape: The functional state of the outer-membrane β-barrel protein OmpG molds its unfolding energy landscape

Quantifying interactions that switch the β-barrel membrane protein OmpG from the open to the closed state

Probing the interactions of carboxy-atractyloside and atractyloside with the yeast mitochondrial ADP/ATP carrier

Molecular Force Spectroscopy

Further reading

YidC assists the stepwise and stochastic folding of membrane proteins

Molecular plasticity of the human voltage-dependent anion channel embedded into a membrane

Quantifying and identifying two ligand-binding sites while imaging native human membrane receptors by AFM

Impact of holdase chaperones Skp and SurA on the folding of β-barrel outer membrane proteins

Single-molecule force spectroscopy of membrane proteins from membranes freely spanning across nanoscopic pores

Dynamic single-molecule force spectroscopy of rhodopsin in native membranes

Observing a lipid-dependent alteration in single lactose permeases

Action of the Hsp70 chaperone system observed with single proteins

Substrate-induced changes in the structural properties of LacY

The peptide transporter DtpA populates two alternate conformations from which inhibitor binding promotes one

Mechanistic explanation of different unfolding behaviors observed for transmembrane and soluble β-barrel proteins

Single-molecule force spectroscopy of G-protein coupled receptors

Nanomechanical properties of proteins and membranes depend on loading rate and electrostatic interactions

Structural, kinetic, and mechanical differences between dark-state rhodopsin and opsin

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

Out but not in: β-strands shape the unfolding pathway but not the refolding of the large transmembrane β-barrel protein FhuA

Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase

Ligand-specific interactions modulate kinetic, energetic and mechanical properties of the human β2 adrenergic receptor

Structural, energetic and mechanical perturbations in a rhodopsin mutant that causes congenital stationary night blindness

Single-molecule force spectroscopy from nanodiscs – an assay to quantify folding, stability and interactions of native membrane proteins

KpOmpA a transmembrane protein anchoring the outer membrane of Klebsiella pneumoniae unfolds and refolds in response to tensile load

Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+-coupled betaine symporter BetP

Competing interactions stabilize pro- and anti-aggregant conformations of human Tau

One β-hairpin after the other: Exploring refolding pathways and kinetics of the transmembrane β-barrel protein OmpG

High-resolution atomic force microscopy and spectroscopy of native membrane proteins

Five challenges to bringing single-molecule force spectroscopy into the living cell

Conservation of molecular interactions stabilizing bovine and mouse rhodopsin

A ”force buffer” protecting immunoglobulin titin

Assessing the structure and function of single biomolecules with scanning transmission electron and atomic force microscopes

Dual energy landscape: The functional state of the outer-membrane β-barrel protein OmpG molds its unfolding energy landscape

Quantifying interactions that switch the β-barrel membrane protein OmpG from the open to the closed state

Probing the interactions of carboxy-atractyloside and atractyloside with the yeast mitochondrial ADP/ATP carrier

Locating an extracellular K⁺-dependent interaction site that modulates betaine-binding of the Na⁺-coupled betaine symporter BetP