@article{22917de0363d4d9fa2a21bfb551aad2d,
title = "Stoichiometric Analyses of Soluble CD4 to Native-like HIV-1 Envelope by Single-Molecule Fluorescence Spectroscopy",
abstract = "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.",
keywords = "HIV-1 Env trimers, association kinetics, cooperativity, ligand binding, single-molecule fluorescence, soluble CD4, stoichiometric analyses",
author = "Parul Agrawal and DeVico, {Anthony L.} and Foulke, {James S.} and Lewis, {George K.} and Marzena Pazgier and Krishanu Ray",
note = "Funding Information: Research reported in this publication was supported by the National Institute of General Medical Sciences and National Institute of Allergy and Infectious Diseases of the NIH under awards numbers R01 GM117836, R01 AI150447, and R01GM117836-S1 to K.R.; R01 AI116274 and R01 AI129769 to M.P.; and P01 AI120756 to A.L.D. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We thank Dr. John Moore of Cornell University for the generous gift of BG505 SOSIP.664 trimers. We thank Amir Dashti and Panjamurthy Kuppusamy for technical assistance. We greatly appreciate Vivek M. Agrawal for his help with creating Python-based scripts to automate step tracing from intensity-time trajectories using rule-based processing. P.A. performed experiments, contributed to new reagents, analyzed data, and wrote the manuscript. A.L.D. contributed to new reagents, analyzed data, and edited the manuscript. J.S.F. contributed to new reagents. G.K.L. analyzed data and edited the manuscript. M.P. performed structural modeling. K.R. designed and performed research, analyzed data, and wrote the manuscript. The authors declare no competing interests. Funding Information: Research reported in this publication was supported by the National Institute of General Medical Sciences and National Institute of Allergy and Infectious Diseases of the NIH under awards numbers R01 GM117836 , R01 AI150447 , and R01GM117836-S1 to K.R.; R01 AI116274 and R01 AI129769 to M.P.; and P01 AI120756 to A.L.D. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We thank Dr. John Moore of Cornell University for the generous gift of BG505 SOSIP.664 trimers. We thank Amir Dashti and Panjamurthy Kuppusamy for technical assistance. We greatly appreciate Vivek M. Agrawal for his help with creating Python-based scripts to automate step tracing from intensity-time trajectories using rule-based processing. Publisher Copyright: {\textcopyright} 2019 The Authors",
year = "2019",
month = oct,
day = "1",
doi = "10.1016/j.celrep.2019.08.074",
language = "English",
volume = "29",
pages = "176--186.e4",
journal = "Cell Reports",
issn = "2211-1247",
number = "1",
}