Stoichiometric Analyses of Soluble CD4 to Native-like HIV-1 Envelope by Single-Molecule Fluorescence Spectroscopy

Parul Agrawal, Anthony L. DeVico, James S. Foulke, George K. Lewis, Marzena Pazgier, Krishanu Ray*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


Analyses of HIV-1 envelope (Env) binding to CD4, and the conformational changes the interactions induce, inform the molecular mechanisms and factors governing HIV-1 infection. To address these questions, we used a single-molecule detection (SMD) approach to study the nature of reactions between soluble CD4 (sCD4) and soluble HIV-1 trimers. SMD of these reactions distinguished a mixture of one, two, or three CD4-bound trimer species. Single-ligand trimers were favored at early reaction times and ligand-saturated trimers later. Furthermore, some trimers occupied by one sCD4 molecule did not bind additional ligands, whereas the majority of two ligand-bound species rapidly transitioned to the saturated state. Quantification of liganded trimers observed in reactions with various sCD4 concentrations reflected an overall negative cooperativity in ligand binding. Collectively, our results highlight the general utility of SMD in studying protein interactions and provide critical insights on the nature of sCD4-HIV-1 Env interactions.

Original languageEnglish
Pages (from-to)176-186.e4
JournalCell Reports
Issue number1
StatePublished - 1 Oct 2019
Externally publishedYes


  • HIV-1 Env trimers
  • association kinetics
  • cooperativity
  • ligand binding
  • single-molecule fluorescence
  • soluble CD4
  • stoichiometric analyses


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