As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation, and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies, but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C-433C (DS) variant, specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like particle and soluble formats providing a new generation of vaccine antigens.

SummaryNeurotransmitter:Na+ symporters (NSS) remove neurotransmitters from the synapse in a reuptake process driven by the Na+ gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behavior and mood. In order to probe the nature of conformational changes associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay, in combination with functional and computational studies, using the prokaryotic NSS homolog LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding and/or transport. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that may be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism coupling ion and substrate binding to transport.

Fluorescence applications requiring high photostability often depend on the use of solution additives to enhance fluorophore performance. Here we demonstrate that the direct or proximal conjugation of cyclooctatetraene (COT), 4-nitrobenzyl alcohol (NBA) or Trolox to the cyanine fluorophore Cy5 dramatically enhanced fluorophore photostability without otherwise affecting its native spectral characteristics. Such conjugation is a powerful means of improving the robustness of fluorescence-based applications demanding long-lived, nonblinking fluorescence emission.

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