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Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM.

Pfreundschuh M, Alsteens D, Wieneke R, Zhang C, Coughlin SR, Tampé R, Kobilka BK, Müller DJ - Nat Commun (2015)

Bottom Line: Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands.Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1.We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH), Mattenstrasse 26, 4058 Basel, Switzerland.

ABSTRACT
A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

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Imaging PAR1 proteoliposomes using bifunctionalized AFM tips and mapping the specific binding of SFLLRN or of tris-NTA ligands.(a–d) Imaging the proteoliposome and mapping the binding of the SFLLRN ligand to PAR1. (a) AFM topograph of a PAR1 proteoliposome recorded with inactivated tris-NTA (tris-NTA(−)) and active SFLLRN (SFLLRN(+)) ligand. To prevent tris-NTA interacting with the His10-tag of PAR1, experiments were conducted in the absence of Ni2+. (b) Adhesion map showing specific rupture forces on the proteoliposome (dashed) imaged in a. (c) FD curves recording specific interactions in b at distances corresponding to the length of the stretched PEG linker tethering the SFLLRN ligand to the AFM tip. (d) Rupture forces of single SFLLRN–PAR1 bonds range from 35 to 80 pN. (e–h) Imaging the proteoliposome and mapping the binding of the tris-NTA ligand to the His10-tag of PAR1. (e) AFM topograph of the PAR1 proteoliposome recorded with activated tris-NTA (tris-NTA(+)) and blocked SFLLRN (SFLLRN(−)) ligand. To promote tris-NTA binding to the His10-tag and to prevent the SFLLRN ligand binding to PAR1 the experiments were conducted in the presence of Ni2+ and the antagonist BMS. (f) Adhesion map showing specific rupture forces on the proteoliposome (dashed) imaged in e. (g) FD curves recording specific interactions in f. (h) Rupture forces of single tris-NTA–His10-tag bonds range from 65 to 140 pN. The encircled positions of AFM topographs and adhesion maps indicate where specific interactions were detected. Topographic heights (a and e) are indicated by colour bars. Rupture force distributions (c,d,g,h) shown at 5 pN bin size. Images were recorded in 300 mM NaCl, 20 mM HEPES, 25 mM MgCl2, pH 7.2 and if stated 2 μM BMS or/and 5 mM NiCl2. Experiments were repeated six times, each time we prepared a new sample and used different functionalized AFM tips. Scale bars, 500 nm (a,e).
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f2: Imaging PAR1 proteoliposomes using bifunctionalized AFM tips and mapping the specific binding of SFLLRN or of tris-NTA ligands.(a–d) Imaging the proteoliposome and mapping the binding of the SFLLRN ligand to PAR1. (a) AFM topograph of a PAR1 proteoliposome recorded with inactivated tris-NTA (tris-NTA(−)) and active SFLLRN (SFLLRN(+)) ligand. To prevent tris-NTA interacting with the His10-tag of PAR1, experiments were conducted in the absence of Ni2+. (b) Adhesion map showing specific rupture forces on the proteoliposome (dashed) imaged in a. (c) FD curves recording specific interactions in b at distances corresponding to the length of the stretched PEG linker tethering the SFLLRN ligand to the AFM tip. (d) Rupture forces of single SFLLRN–PAR1 bonds range from 35 to 80 pN. (e–h) Imaging the proteoliposome and mapping the binding of the tris-NTA ligand to the His10-tag of PAR1. (e) AFM topograph of the PAR1 proteoliposome recorded with activated tris-NTA (tris-NTA(+)) and blocked SFLLRN (SFLLRN(−)) ligand. To promote tris-NTA binding to the His10-tag and to prevent the SFLLRN ligand binding to PAR1 the experiments were conducted in the presence of Ni2+ and the antagonist BMS. (f) Adhesion map showing specific rupture forces on the proteoliposome (dashed) imaged in e. (g) FD curves recording specific interactions in f. (h) Rupture forces of single tris-NTA–His10-tag bonds range from 65 to 140 pN. The encircled positions of AFM topographs and adhesion maps indicate where specific interactions were detected. Topographic heights (a and e) are indicated by colour bars. Rupture force distributions (c,d,g,h) shown at 5 pN bin size. Images were recorded in 300 mM NaCl, 20 mM HEPES, 25 mM MgCl2, pH 7.2 and if stated 2 μM BMS or/and 5 mM NiCl2. Experiments were repeated six times, each time we prepared a new sample and used different functionalized AFM tips. Scale bars, 500 nm (a,e).

Mentions: After bifunctionalizing the AFM tip, we adsorbed PAR1 proteoliposomes to freshly cleaved mica and imaged the sample in buffer solution by FD-based AFM using the bifunctionalized tip (Fig. 2). To first detect only the interaction of the SFLLRN ligand to PAR1, we used imaging buffer lacking Ni2+ ions, which are needed to coordinate the tris-NTA to the His10-tag. We recorded FD-based AFM topographs with 256 × 256 pixels (2.5 × 2.5 μm2) and one approach and one retraction FD curve per pixel. The exemplified topograph precisely showed the lipid membrane with protrusions corresponding to single and assemblies of several PAR1s (Fig. 2a and Supplementary Fig. 2). The single-layered lipid membrane indicated that on adsorption to mica the proteoliposome opened. Analysing the adhesion detected in each FD curve recorded in the topograph allowed to calculate the adhesion map (Fig. 2b). Correlation of this adhesion map with the topograph localized unspecific interactions of the AFM tip with the supporting mica and specific interactions with the receptors. Characteristic adhesive forces arising from specific interactions were detected at tip-sample distances >7 nm, which correlates to the length of the extended PEG27 linker (≈10 nm) and of the 28-aa-long native N-terminal sequence of PAR1 tethering the SFLLRN ligand to the tip (Fig. 2c and Supplementary Note 1). The variation of the distances at which the rupture forces were detected resulted from SFFLRN ligands being attached at different positions to the AFM tip (Supplementary Fig. 3). In rare cases (<5%), rupture forces were detected at distances longer than the linker system, suggesting that the mechanical force applied to the receptor lifted the soft lipid membrane29. Such force curves were excluded from analysis (Supplementary Note 1). As a further control, we validated the extension profile of every single-adhesion peak detected in a FD curve by fitting the polyethylene glycol (PEG)-polypeptide linker system attaching the SFLLRN ligand to the AFM tip (Supplementary Figs 3,4 and 7). The forces of these adhesion peaks ranged from 35 to 80 pN (Fig. 2d), which is the typical force required to separate ligand–receptor bonds30313233. In several controls, we showed that non-functionalized AFM tips, AFM tips functionalized with only the PEG27 spacer and AFM tips functionalized with the scrambled SFLLRN peptide LFRLSN did not detect specific interactions with the supporting mica or with the PAR1 proteoliposome (Supplementary Figs 5 and 6).


Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM.

Pfreundschuh M, Alsteens D, Wieneke R, Zhang C, Coughlin SR, Tampé R, Kobilka BK, Müller DJ - Nat Commun (2015)

Imaging PAR1 proteoliposomes using bifunctionalized AFM tips and mapping the specific binding of SFLLRN or of tris-NTA ligands.(a–d) Imaging the proteoliposome and mapping the binding of the SFLLRN ligand to PAR1. (a) AFM topograph of a PAR1 proteoliposome recorded with inactivated tris-NTA (tris-NTA(−)) and active SFLLRN (SFLLRN(+)) ligand. To prevent tris-NTA interacting with the His10-tag of PAR1, experiments were conducted in the absence of Ni2+. (b) Adhesion map showing specific rupture forces on the proteoliposome (dashed) imaged in a. (c) FD curves recording specific interactions in b at distances corresponding to the length of the stretched PEG linker tethering the SFLLRN ligand to the AFM tip. (d) Rupture forces of single SFLLRN–PAR1 bonds range from 35 to 80 pN. (e–h) Imaging the proteoliposome and mapping the binding of the tris-NTA ligand to the His10-tag of PAR1. (e) AFM topograph of the PAR1 proteoliposome recorded with activated tris-NTA (tris-NTA(+)) and blocked SFLLRN (SFLLRN(−)) ligand. To promote tris-NTA binding to the His10-tag and to prevent the SFLLRN ligand binding to PAR1 the experiments were conducted in the presence of Ni2+ and the antagonist BMS. (f) Adhesion map showing specific rupture forces on the proteoliposome (dashed) imaged in e. (g) FD curves recording specific interactions in f. (h) Rupture forces of single tris-NTA–His10-tag bonds range from 65 to 140 pN. The encircled positions of AFM topographs and adhesion maps indicate where specific interactions were detected. Topographic heights (a and e) are indicated by colour bars. Rupture force distributions (c,d,g,h) shown at 5 pN bin size. Images were recorded in 300 mM NaCl, 20 mM HEPES, 25 mM MgCl2, pH 7.2 and if stated 2 μM BMS or/and 5 mM NiCl2. Experiments were repeated six times, each time we prepared a new sample and used different functionalized AFM tips. Scale bars, 500 nm (a,e).
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f2: Imaging PAR1 proteoliposomes using bifunctionalized AFM tips and mapping the specific binding of SFLLRN or of tris-NTA ligands.(a–d) Imaging the proteoliposome and mapping the binding of the SFLLRN ligand to PAR1. (a) AFM topograph of a PAR1 proteoliposome recorded with inactivated tris-NTA (tris-NTA(−)) and active SFLLRN (SFLLRN(+)) ligand. To prevent tris-NTA interacting with the His10-tag of PAR1, experiments were conducted in the absence of Ni2+. (b) Adhesion map showing specific rupture forces on the proteoliposome (dashed) imaged in a. (c) FD curves recording specific interactions in b at distances corresponding to the length of the stretched PEG linker tethering the SFLLRN ligand to the AFM tip. (d) Rupture forces of single SFLLRN–PAR1 bonds range from 35 to 80 pN. (e–h) Imaging the proteoliposome and mapping the binding of the tris-NTA ligand to the His10-tag of PAR1. (e) AFM topograph of the PAR1 proteoliposome recorded with activated tris-NTA (tris-NTA(+)) and blocked SFLLRN (SFLLRN(−)) ligand. To promote tris-NTA binding to the His10-tag and to prevent the SFLLRN ligand binding to PAR1 the experiments were conducted in the presence of Ni2+ and the antagonist BMS. (f) Adhesion map showing specific rupture forces on the proteoliposome (dashed) imaged in e. (g) FD curves recording specific interactions in f. (h) Rupture forces of single tris-NTA–His10-tag bonds range from 65 to 140 pN. The encircled positions of AFM topographs and adhesion maps indicate where specific interactions were detected. Topographic heights (a and e) are indicated by colour bars. Rupture force distributions (c,d,g,h) shown at 5 pN bin size. Images were recorded in 300 mM NaCl, 20 mM HEPES, 25 mM MgCl2, pH 7.2 and if stated 2 μM BMS or/and 5 mM NiCl2. Experiments were repeated six times, each time we prepared a new sample and used different functionalized AFM tips. Scale bars, 500 nm (a,e).
Mentions: After bifunctionalizing the AFM tip, we adsorbed PAR1 proteoliposomes to freshly cleaved mica and imaged the sample in buffer solution by FD-based AFM using the bifunctionalized tip (Fig. 2). To first detect only the interaction of the SFLLRN ligand to PAR1, we used imaging buffer lacking Ni2+ ions, which are needed to coordinate the tris-NTA to the His10-tag. We recorded FD-based AFM topographs with 256 × 256 pixels (2.5 × 2.5 μm2) and one approach and one retraction FD curve per pixel. The exemplified topograph precisely showed the lipid membrane with protrusions corresponding to single and assemblies of several PAR1s (Fig. 2a and Supplementary Fig. 2). The single-layered lipid membrane indicated that on adsorption to mica the proteoliposome opened. Analysing the adhesion detected in each FD curve recorded in the topograph allowed to calculate the adhesion map (Fig. 2b). Correlation of this adhesion map with the topograph localized unspecific interactions of the AFM tip with the supporting mica and specific interactions with the receptors. Characteristic adhesive forces arising from specific interactions were detected at tip-sample distances >7 nm, which correlates to the length of the extended PEG27 linker (≈10 nm) and of the 28-aa-long native N-terminal sequence of PAR1 tethering the SFLLRN ligand to the tip (Fig. 2c and Supplementary Note 1). The variation of the distances at which the rupture forces were detected resulted from SFFLRN ligands being attached at different positions to the AFM tip (Supplementary Fig. 3). In rare cases (<5%), rupture forces were detected at distances longer than the linker system, suggesting that the mechanical force applied to the receptor lifted the soft lipid membrane29. Such force curves were excluded from analysis (Supplementary Note 1). As a further control, we validated the extension profile of every single-adhesion peak detected in a FD curve by fitting the polyethylene glycol (PEG)-polypeptide linker system attaching the SFLLRN ligand to the AFM tip (Supplementary Figs 3,4 and 7). The forces of these adhesion peaks ranged from 35 to 80 pN (Fig. 2d), which is the typical force required to separate ligand–receptor bonds30313233. In several controls, we showed that non-functionalized AFM tips, AFM tips functionalized with only the PEG27 spacer and AFM tips functionalized with the scrambled SFLLRN peptide LFRLSN did not detect specific interactions with the supporting mica or with the PAR1 proteoliposome (Supplementary Figs 5 and 6).

Bottom Line: Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands.Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1.We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH), Mattenstrasse 26, 4058 Basel, Switzerland.

ABSTRACT
A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

Show MeSH
Related in: MedlinePlus