Observing the Folding of Single Proteins into Membranes

One of the great challenges for molecular and cell biologists is to understand how a polypeptide defines the three-dimensional structure of the a protein. It is even more difficult to understand this problem for membrane proteins because so little was known about what they looked like. In the recent years the situation has improved markedly as more membrane protein strutcures have been solved. However, it remains still largely unknown how a polypeptide itself or with the help of chaperones, translocons and insertases can insert and fold into the native membrane. We thus develop methods that allow to observe the unfolding and folding of single polypeptides into membranes at native-like conditions. Currently the resolution of these folding experiments allow us to observe the unfolding steps and folding steps of single secondary structures and how theses steps form the folding pathway towards the completely folded functional membrane protein.

Enlarged view: Folding pathways and free-energy landscape of FhuA receptors — Folding pathways and free-energy landscape of FhuA receptors. (a) Insertion and folding pathways of FhuA in the absence of chaperones and in the presence of SurA (orange). Without chaperones, the majority of unfolded FhuA receptors misfold. The misfolded polypeptide is assumed to locate inside and/or outside the membrane. SurA, when present, stabilizes the unfolded state of FhuA and promotes stepwise insertion and folding of β-hairpins in the lipid membrane. This stepwise insertion of secondary structures proceeds until the substrate completes the folding of the receptor. (b) Hypothetical folding free-energy landscape of FhuA in the presence of SurA. SurA (orange) is spatially excluded from the lipid membrane (blue). Each β-hairpin inserted into the lipid membrane is stabilized by a free-energy well. The lipid membrane thus acts as a free-energy sink for the insertion of β-hairpins and physically separates transient folds from the chaperones. Figure and legend adapted from J. Thoma *et al.* Nat. Struct. Mol. Biol. (2015) 22, 795-802.

Observing the Folding of Single Proteins into Membranes

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

Monitoring backbone hydrogen bond formation in β-barrel membrane protein folding

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

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

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

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

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

Conservation of molecular interactions stabilizing bovine and mouse rhodopsin

A ”force buffer” protecting immunoglobulin titin

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

One β-hairpin after the other: Exploring mechanical unfolding pathways of the transmembrane β-barrel protein OmpG

Modulation of molecular interactions and function by rhodopsin palmitylation

Substrate binding tunes conformational flexibility and kinetic stability of an amino acid transporter

AFM: A nanotool in membrane biology

High-throughput single-molecule force spectroscopy for membrane proteins

Fully automated single-molecule force spectroscopy for screening applications

Transducer binding establishes localized interactions that tune sensory rhodopsin II

Role of extracellular glutamic acids in the stability and energy landscape of bacteriorhodopsin

From valleys to ridges: Exploring the energy landscape of single membrane proteins

Folding and assembly of proteorhodopsin

Mechanical properties of bovine rhodopsin and bacteriorhodopsin dictate energy landscape and guide conformational changes

Point mutations of membrane protein alter energy landscape and unfolding pathways

Examining the dynamic energy landscape of an antiporter upon inhibitor binding

Atomic force microscopy and spectroscopy of native membrane proteins

Free energy of membrane protein unfolding derived from single-molecule force measurements

Stabilizing effect of Zn2+ in native bovine rhodopsin

Transmembrane helices have rough energy surfaces

Detecting molecular interactions that stabilize, activate and guide ligand-binding of the sodium/proton antiporter MjNhaP1 from Methanococcus jannaschii

Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy

Differentiating ligand and inhibitor interactions of a single antiporter

Bacteriorhodopsin folds into the membrane against an external force

Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy

Direct measurement of single-molecule visco-elasticity in AFM force-extension measurements

Observing folding pathways and kinetics of a single membrane protein

Locating ligand binding and activation of a single antiporter

Complex stability of single proteins explored by forced unfolding experiments

Probing different origins of potential barriers stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin

Dynamic response of single bacteriorhodopsins, determined by single-molecule force modulation spectroscopy

Probing the energy landscape of the membrane protein bacteriorhodopsin

Controlled unfolding and refolding of a single sodium-proton antiporter

Temperature dependent unfolding of bacteriorhodopsin

Determining molecular forces that stabilize human aquaporin-1

Stability of bacteriorhodopsin α-helices and loops analyzed by single molecule force spectroscopy

Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin

Unfolding pathways of individual bacteriorhodopsins

Stabilizing effect of Zn²⁺ in native bovine rhodopsin