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Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein – Ligand Interactions

View Article: PubMed Central - PubMed

ABSTRACT

A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

No MeSH data available.


Related in: MedlinePlus

A–E) Acceptor direct excitation background‐corrected fluorescence spectra of QD‐PEGn‐Man (λEM=554 nm, final CQD=40 nm) after binding to Atto‐594‐labeled proteins: A) DC‐SIGN+QD‐PEG13‐Man; B) DC‐SIGN+QD‐EG3‐Man; C) DC‐SIGNR+QD‐PEG13‐Man; D) DC‐SIGNR+QD‐EG3‐Man; E) DC‐SIGN CRD monomer+QD‐EG3‐Man. F) Relationship between the apparent FRET ratio (I626/I554) and protein concentration fitted to the Hill equation. G,H) Luciferase activities of cell lysates of DC‐SIGN/R expressing 293T cells after exposure to an Ebola virus glycoprotein‐bearing, luciferase‐encoding murine leukemia virus (MLV‐EBOV‐GP) vector in the presence of indicated amounts of QD‐EG3‐Man in Dulbecco's modified eagle medium supplemented with 10 % fetal bovine serum. A MLV vector bearing the vesicular stomatitis virus glycoprotein (MLV‐VSV‐G) was used as negative control. Cells transfected with empty plasmid (pcDNA) were used as additional negative controls. In panel (G), the reduction of EBOV‐GP‐dependent transduction by 250 nm QD‐EG3‐Man was statistically significant from the 0 nm QD control (p=0.024).
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anie201600593-fig-0002: A–E) Acceptor direct excitation background‐corrected fluorescence spectra of QD‐PEGn‐Man (λEM=554 nm, final CQD=40 nm) after binding to Atto‐594‐labeled proteins: A) DC‐SIGN+QD‐PEG13‐Man; B) DC‐SIGN+QD‐EG3‐Man; C) DC‐SIGNR+QD‐PEG13‐Man; D) DC‐SIGNR+QD‐EG3‐Man; E) DC‐SIGN CRD monomer+QD‐EG3‐Man. F) Relationship between the apparent FRET ratio (I626/I554) and protein concentration fitted to the Hill equation. G,H) Luciferase activities of cell lysates of DC‐SIGN/R expressing 293T cells after exposure to an Ebola virus glycoprotein‐bearing, luciferase‐encoding murine leukemia virus (MLV‐EBOV‐GP) vector in the presence of indicated amounts of QD‐EG3‐Man in Dulbecco's modified eagle medium supplemented with 10 % fetal bovine serum. A MLV vector bearing the vesicular stomatitis virus glycoprotein (MLV‐VSV‐G) was used as negative control. Cells transfected with empty plasmid (pcDNA) were used as additional negative controls. In panel (G), the reduction of EBOV‐GP‐dependent transduction by 250 nm QD‐EG3‐Man was statistically significant from the 0 nm QD control (p=0.024).

Mentions: To probe the multivalent binding by FRET, DC‐SIGN was labeled with Atto‐594 dye (Section A51/52) on a site‐specifically introduced cysteine residue. The dye labeling did not affect its specific binding to a Sepharose‐mannose column. The Atto‐594‐QD FRET pair had a respectable Förster radius (R0=4.7/5.0 nm for QD‐EG3‐Man/QD‐PEG13‐Man, respectively; see Figure S2). Binding of the labeled DC‐SIGN to the QDs yielded significantly reduced QD fluorescence at 554 nm together with concurrently enhanced Atto‐594 FRET signal at 626 nm (Figure 2 A,B), which was fully consistent with a QD‐sensitized Atto‐594 FRET mechanism. Stronger FRET signals and more severely quenched QD fluorescence were observed for DC‐SIGN binding to QD‐EG3‐Man over QD‐PEG13‐Man, indicating more efficient FRET in the former pair. Both bindings displayed excellent fits (R2>0.99) by the single QD donor FRET with N identical acceptors model,2 yielding QD–dye distances (r) of about 6.8/9.8 nm for QD‐EG3‐Man/QD‐PEG13‐Man, respectively (Figure S3). These r values roughly matched the sum of QD core radius plus respective fully extended ligand length (ca. 6.5 and 10.0 nm; Figure S1).


Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein – Ligand Interactions
A–E) Acceptor direct excitation background‐corrected fluorescence spectra of QD‐PEGn‐Man (λEM=554 nm, final CQD=40 nm) after binding to Atto‐594‐labeled proteins: A) DC‐SIGN+QD‐PEG13‐Man; B) DC‐SIGN+QD‐EG3‐Man; C) DC‐SIGNR+QD‐PEG13‐Man; D) DC‐SIGNR+QD‐EG3‐Man; E) DC‐SIGN CRD monomer+QD‐EG3‐Man. F) Relationship between the apparent FRET ratio (I626/I554) and protein concentration fitted to the Hill equation. G,H) Luciferase activities of cell lysates of DC‐SIGN/R expressing 293T cells after exposure to an Ebola virus glycoprotein‐bearing, luciferase‐encoding murine leukemia virus (MLV‐EBOV‐GP) vector in the presence of indicated amounts of QD‐EG3‐Man in Dulbecco's modified eagle medium supplemented with 10 % fetal bovine serum. A MLV vector bearing the vesicular stomatitis virus glycoprotein (MLV‐VSV‐G) was used as negative control. Cells transfected with empty plasmid (pcDNA) were used as additional negative controls. In panel (G), the reduction of EBOV‐GP‐dependent transduction by 250 nm QD‐EG3‐Man was statistically significant from the 0 nm QD control (p=0.024).
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anie201600593-fig-0002: A–E) Acceptor direct excitation background‐corrected fluorescence spectra of QD‐PEGn‐Man (λEM=554 nm, final CQD=40 nm) after binding to Atto‐594‐labeled proteins: A) DC‐SIGN+QD‐PEG13‐Man; B) DC‐SIGN+QD‐EG3‐Man; C) DC‐SIGNR+QD‐PEG13‐Man; D) DC‐SIGNR+QD‐EG3‐Man; E) DC‐SIGN CRD monomer+QD‐EG3‐Man. F) Relationship between the apparent FRET ratio (I626/I554) and protein concentration fitted to the Hill equation. G,H) Luciferase activities of cell lysates of DC‐SIGN/R expressing 293T cells after exposure to an Ebola virus glycoprotein‐bearing, luciferase‐encoding murine leukemia virus (MLV‐EBOV‐GP) vector in the presence of indicated amounts of QD‐EG3‐Man in Dulbecco's modified eagle medium supplemented with 10 % fetal bovine serum. A MLV vector bearing the vesicular stomatitis virus glycoprotein (MLV‐VSV‐G) was used as negative control. Cells transfected with empty plasmid (pcDNA) were used as additional negative controls. In panel (G), the reduction of EBOV‐GP‐dependent transduction by 250 nm QD‐EG3‐Man was statistically significant from the 0 nm QD control (p=0.024).
Mentions: To probe the multivalent binding by FRET, DC‐SIGN was labeled with Atto‐594 dye (Section A51/52) on a site‐specifically introduced cysteine residue. The dye labeling did not affect its specific binding to a Sepharose‐mannose column. The Atto‐594‐QD FRET pair had a respectable Förster radius (R0=4.7/5.0 nm for QD‐EG3‐Man/QD‐PEG13‐Man, respectively; see Figure S2). Binding of the labeled DC‐SIGN to the QDs yielded significantly reduced QD fluorescence at 554 nm together with concurrently enhanced Atto‐594 FRET signal at 626 nm (Figure 2 A,B), which was fully consistent with a QD‐sensitized Atto‐594 FRET mechanism. Stronger FRET signals and more severely quenched QD fluorescence were observed for DC‐SIGN binding to QD‐EG3‐Man over QD‐PEG13‐Man, indicating more efficient FRET in the former pair. Both bindings displayed excellent fits (R2>0.99) by the single QD donor FRET with N identical acceptors model,2 yielding QD–dye distances (r) of about 6.8/9.8 nm for QD‐EG3‐Man/QD‐PEG13‐Man, respectively (Figure S3). These r values roughly matched the sum of QD core radius plus respective fully extended ligand length (ca. 6.5 and 10.0 nm; Figure S1).

View Article: PubMed Central - PubMed

ABSTRACT

A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

No MeSH data available.


Related in: MedlinePlus