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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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AFM mapping of two different ligand-binding events using a chemically bifunctionalized AFM tip.(a) Mapping adhesion forces on membrane proteins using FD-based AFM. When recording an AFM topograph an approach (blue) and retraction (red) cycle between AFM tip and biological sample is performed for every pixel of the image. In each cycle, the cantilever deflection (for example, force) and the distance travelled by the AFM tip is monitored and transformed into an approach and retraction FD curve (Supplementary Fig. 1). (b) Bifunctionalization of the AFM tip. The amino-functionalized Si3N4 AFM tip is functionalized by hetero-bifunctional N-hydroxysuccinimide-PEG27-maleimide linkers to which the thiol bearing thrombin receptor-activating peptide (TRAP) and Ni2+-loaded tris-NTA (tris-Ni2+-NTA) are bound. (c) The membrane-embedded PAR1 reveals an intracellular C-terminal His10-tag (green) and an extracellular TRAP (here the native SFLLRN peptide ligand)-binding pocket (red), which can specifically bind the tris-Ni2+-NTA and the SFLLRN ligands, respectively.
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f1: AFM mapping of two different ligand-binding events using a chemically bifunctionalized AFM tip.(a) Mapping adhesion forces on membrane proteins using FD-based AFM. When recording an AFM topograph an approach (blue) and retraction (red) cycle between AFM tip and biological sample is performed for every pixel of the image. In each cycle, the cantilever deflection (for example, force) and the distance travelled by the AFM tip is monitored and transformed into an approach and retraction FD curve (Supplementary Fig. 1). (b) Bifunctionalization of the AFM tip. The amino-functionalized Si3N4 AFM tip is functionalized by hetero-bifunctional N-hydroxysuccinimide-PEG27-maleimide linkers to which the thiol bearing thrombin receptor-activating peptide (TRAP) and Ni2+-loaded tris-NTA (tris-Ni2+-NTA) are bound. (c) The membrane-embedded PAR1 reveals an intracellular C-terminal His10-tag (green) and an extracellular TRAP (here the native SFLLRN peptide ligand)-binding pocket (red), which can specifically bind the tris-Ni2+-NTA and the SFLLRN ligands, respectively.

Mentions: To detect specific interactions of a biological sample FD-based AFM contours the sample topography while recording for every topographic pixel at least one FD curve (Supplementary Fig. 1). On functionalizing the AFM tip with one ligand it has been shown that FD-based AFM can detect the binding events of the ligand and localize these specific interactions to the topography of the protein12. In our approach we wanted to contour the topography of human PAR1 proteoliposomes and to simultaneously detect the interaction of the sample with two different ligands (Fig. 1a). Thus, to simultaneously detect the binding of PAR1 to extracellular and intracellular ligands by FD-based AFM, we developed a procedure to bifunctionalize the AFM tip with two different ligands (Fig. 1b). One ligand was the native SFLLRN ligand of PAR1. To mimic as closely as possible the native binding of the ligand to PAR1 we left the SFLLRN peptide at the 28-amino acid (aa)-long N-terminal end of thrombin-cleaved PAR1. The other ligand was a tris-NTA group, which in presence of Ni2+ ions can interact with a His10-tag fused to the intracellular C terminus of the receptor (Fig. 1b–c). Whereas the tris-NTA group shows an affinity Kd to bind the His10-tag of ≈10–30 nM, the SFLLRN ligand shows an affinity for binding PAR1 of ≈200–800 nM (refs 24, 25, 26). To separate unspecific from specific interactions both ligands were covalently attached to a previously amino-functionalized AFM tip via a ≈10-nm-long PEG27 spacer2728.


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)

AFM mapping of two different ligand-binding events using a chemically bifunctionalized AFM tip.(a) Mapping adhesion forces on membrane proteins using FD-based AFM. When recording an AFM topograph an approach (blue) and retraction (red) cycle between AFM tip and biological sample is performed for every pixel of the image. In each cycle, the cantilever deflection (for example, force) and the distance travelled by the AFM tip is monitored and transformed into an approach and retraction FD curve (Supplementary Fig. 1). (b) Bifunctionalization of the AFM tip. The amino-functionalized Si3N4 AFM tip is functionalized by hetero-bifunctional N-hydroxysuccinimide-PEG27-maleimide linkers to which the thiol bearing thrombin receptor-activating peptide (TRAP) and Ni2+-loaded tris-NTA (tris-Ni2+-NTA) are bound. (c) The membrane-embedded PAR1 reveals an intracellular C-terminal His10-tag (green) and an extracellular TRAP (here the native SFLLRN peptide ligand)-binding pocket (red), which can specifically bind the tris-Ni2+-NTA and the SFLLRN ligands, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660198&req=5

f1: AFM mapping of two different ligand-binding events using a chemically bifunctionalized AFM tip.(a) Mapping adhesion forces on membrane proteins using FD-based AFM. When recording an AFM topograph an approach (blue) and retraction (red) cycle between AFM tip and biological sample is performed for every pixel of the image. In each cycle, the cantilever deflection (for example, force) and the distance travelled by the AFM tip is monitored and transformed into an approach and retraction FD curve (Supplementary Fig. 1). (b) Bifunctionalization of the AFM tip. The amino-functionalized Si3N4 AFM tip is functionalized by hetero-bifunctional N-hydroxysuccinimide-PEG27-maleimide linkers to which the thiol bearing thrombin receptor-activating peptide (TRAP) and Ni2+-loaded tris-NTA (tris-Ni2+-NTA) are bound. (c) The membrane-embedded PAR1 reveals an intracellular C-terminal His10-tag (green) and an extracellular TRAP (here the native SFLLRN peptide ligand)-binding pocket (red), which can specifically bind the tris-Ni2+-NTA and the SFLLRN ligands, respectively.
Mentions: To detect specific interactions of a biological sample FD-based AFM contours the sample topography while recording for every topographic pixel at least one FD curve (Supplementary Fig. 1). On functionalizing the AFM tip with one ligand it has been shown that FD-based AFM can detect the binding events of the ligand and localize these specific interactions to the topography of the protein12. In our approach we wanted to contour the topography of human PAR1 proteoliposomes and to simultaneously detect the interaction of the sample with two different ligands (Fig. 1a). Thus, to simultaneously detect the binding of PAR1 to extracellular and intracellular ligands by FD-based AFM, we developed a procedure to bifunctionalize the AFM tip with two different ligands (Fig. 1b). One ligand was the native SFLLRN ligand of PAR1. To mimic as closely as possible the native binding of the ligand to PAR1 we left the SFLLRN peptide at the 28-amino acid (aa)-long N-terminal end of thrombin-cleaved PAR1. The other ligand was a tris-NTA group, which in presence of Ni2+ ions can interact with a His10-tag fused to the intracellular C terminus of the receptor (Fig. 1b–c). Whereas the tris-NTA group shows an affinity Kd to bind the His10-tag of ≈10–30 nM, the SFLLRN ligand shows an affinity for binding PAR1 of ≈200–800 nM (refs 24, 25, 26). To separate unspecific from specific interactions both ligands were covalently attached to a previously amino-functionalized AFM tip via a ≈10-nm-long PEG27 spacer2728.

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