Fully automated single-molecule force spectroscopy for screening applications

Struckmeier, Jens; Wahl, R.; Leuschner, M.; Nunes, João M.; Janovjak, Harald; Geisler, Ulrich; Hofmann, G.; Jähnke, Torsten; Müller, Daniel J.

doi:10.1088/0957-4484/19/38/384020

Cited by 32 publications

(24 citation statements)

References 60 publications

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“…Five washing steps followed with the same buffer to remove unbound protein. Refolding experiments were performed on the fully automated AFM 'ForceRobot 300' (JPK Instruments) 40 ARTICLE AFM protocol for automated refolding of 8xI27. To study the refolding of 8xI27 using AFM-based SMFS, we performed refolding experiments comprised by two unfolding segments performed at constant speed (1,200 nm s À 1 ) and separated by a pause segment.…”

Section: Methodsmentioning

confidence: 99%

Action of the Hsp70 chaperone system observed with single proteins

et al. 2015

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In Escherichia coli, the binding of non-native protein substrates to the Hsp70 chaperone DnaK is mediated by the co-chaperone DnaJ. DnaJ accelerates ATP hydrolysis on DnaK, by closing the peptide-binding cleft of DnaK. GrpE catalysed nucleotide exchange and ATP re-binding then lead to substrate release from DnaK, allowing folding. Here we refold immunoglobulin 27 (I27) to better understand how DnaJ-DnaK-GrpE chaperones cooperate. When DnaJ is present, I27 is less likely to misfold and more likely to fold, whereas the unfolded state remains unaffected. Thus, the 'holdase' DnaJ shows foldase behaviour. Misfolding of I27 is fully abrogated when DnaJ cooperates with DnaK, which stabilizes the unfolded state and increases the probability of folding. Addition of GrpE shifts the unfolded fraction of I27 to prechaperone levels. These insights reveal synergistic mechanisms within the evolutionary highly conserved Hsp70 system that prevent substrates from misfolding and promote their productive transition to the native state.

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Section: Methodsmentioning

confidence: 99%

Action of the Hsp70 chaperone system observed with single proteins

et al. 2015

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“…SMFS was carried out using an automated AFM-based SMFS apparatus (ForceRobot 300; JPK Instruments) (74). SMFS data at 5,000 nm/s were recorded using 16-bit data acquisition hardware (NI PCI-6251; National Instruments).…”

Section: Methodsmentioning

confidence: 99%

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

Zocher

Zhang

Rasmussen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

148

182

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The steroid cholesterol is an essential component of eukaryotic membranes, and it functionally modulates membrane proteins, including G protein-coupled receptors. To reveal insight into how cholesterol modulates G protein-coupled receptors, we have used dynamic single-molecule force spectroscopy to quantify the mechanical strength and flexibility, conformational variability, and kinetic and energetic stability of structural segments stabilizing the human β 2 -adrenergic receptor (β 2 AR) in the absence and presence of the cholesterol analog cholesteryl hemisuccinate (CHS). CHS considerably increased the kinetic, energetic, and mechanical stability of almost every structural segment at sufficient magnitude to alter the structure and functional relationship of β 2 AR. One exception was the structural core segment of β 2 AR, which establishes multiple ligand binding sites, and its properties were not significantly influenced by CHS.atomic force microscopy | energy landscape | intermolecular and intramolecular interactions | proteoliposomes

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“…In this respect, SMFS provides insight into the dynamic nature of the conformations that single-peptide transporters assume in the absence and presence of inhibitors. The quickly progressing SMFS methodology soon will allow the interaction forces of membrane proteins to be detected with much improved sensitivity (∼1 pN) and spatial accuracy (∼0.1 nm) and with significantly improved statistics (67,68). In the future these advances may provide much more detailed information about the interactions that stabilize coexisting conformational substates of membrane proteins residing in their physiologically relevant environment.…”

Section: Discussionmentioning

confidence: 99%

Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding

Bippes

Meury

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Peptide transporters (PTRs) of the large PTR family facilitate the uptake of di-and tripeptides to provide cells with amino acids for protein synthesis and for metabolic intermediates. Although several PTRs have been structurally and functionally characterized, how drugs modulate peptide transport remains unclear. To obtain insight into this mechanism, we characterize inhibitor binding to the Escherichia coli PTR dipeptide and tripeptide permease A (DtpA), which shows substrate specificities similar to its human homolog hPEPT1. After demonstrating that Lys[Z-NO 2 ]-Val, the strongest inhibitor of hPEPT1, also acts as a high-affinity inhibitor for DtpA, we used single-molecule force spectroscopy to localize the structural segments stabilizing the peptide transporter and investigated which of these structural segments change stability upon inhibitor binding. This characterization was done with DtpA embedded in the lipid membrane and exposed to physiologically relevant conditions. In the unbound state, DtpA adopts two main alternate conformations in which transmembrane α-helix (TMH) 2 is either stabilized (in ∼43% of DtpA molecules) or not (in ∼57% of DtpA molecules). The two conformations are understood to represent the inward-and outward-facing conformational states of the transporter. With increasing inhibitor concentration, the conformation characterized by a stabilized TMH 2 becomes increasingly prevalent, reaching ∼92% at saturation. Our measurements further suggest that Lys[Z-NO 2 ]-Val interacts with discrete residues in TMH 2 that are important for ligand binding and substrate affinity. These interactions in turn stabilize TMH 2, thereby promoting the inhibited conformation of DtpA.membrane transporter | proton-dependent oligopeptide transporter | major facilitator superfamily | atomic force microscopy | molecular interactions

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Fully automated single-molecule force spectroscopy for screening applications

Cited by 32 publications

References 60 publications

Action of the Hsp70 chaperone system observed with single proteins

Action of the Hsp70 chaperone system observed with single proteins

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

Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding

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Fully automated single-molecule force spectroscopy for screening applications

Cited by 32 publications

References 60 publications

Action of the Hsp70 chaperone system observed with single proteins

Action of the Hsp70 chaperone system observed with single proteins

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

Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding

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Product

Resources

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