The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to spread with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. Here, we generated four neutralizing nanobodies that target the receptor-binding domain of the SARS-CoV-2 spike protein. We defined two distinct binding epitopes using x-ray crystallography and cryo-electron microscopy. Based on the structures, we engineered multivalent nanobodies with more than 100-fold improved neutralizing activity than monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor-binding competition, while other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion, and rendered the virions non-infectious.

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

The photostability of fluorescent labels comprises one of the main limitations in single-molecule fluorescence (SMF) and super-resolution imaging. An attractive strategy to increase the photostability of organic fluorophores relies on their coupling to photostabilizers, e.g., triplet excited state quenchers, rendering self-healing dyes. Herein we report the self-healing properties of trisNTA-Alexa647 fluorophores (NTA, N-nitrilotriacetic acid). Primarily designed to specifically label biomolecules containing an oligohistidine tag, we hypothesized that the increased effective concentration of Ni(II) triplet state quenchers would lead to their improved photostability. We evaluated photon output, survival time, and photon count rate of different Alexa647-labeled trisNTA constructs differing in the length and rigidity of the fluorophore- trisNTA linker. Maximum photon output enhancements of 25-fold versus Alexa647-DNA were recorded for a short tetraproline linker, superseding the solution based photostabilization by Ni(II). Steady-state and time-resolved studies illustrate that trisNTA self-healing role is associated with a dynamic excited triplet state quenching by Ni(II). Here improved photophysical/photochemical properties require for a judicious choice of linker length and rigidity, and in turn a balance between rapid dynamic triplet excited state quenching versus dynamic/static singlet excited state quenching. TrisNTA fluorophores offer superior properties for SMF allowing specific labeling and increased photostability, making them ideal candidates for extended single-molecule imaging techniques.

Small chemical/biological interaction pairs are at the forefront in tracing protein function and interaction at high signal-to-background ratios in cellular pathways. However, the optimal design of scaffold, linker, and chelator head still deserve systematic investigation to achieve the highest affinity and kinetic stability for in vitro and especially cellular applications. We report on a library of N-nitrilotriacetic acid (NTA)-based multivalent chelator heads (MCHs) built on linear, cyclic, and dendritic scaffolds and compare these with regard to their binding affinity and stability for the labeling of cellular His-tagged proteins. Furthermore, we describe a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we outline fundamental differences between the MCH scaffolds and define a cyclic trisNTA chelator that displays the highest affinity and kinetic stability of all reported reversible, low-molecular-weight interaction pairs.