Oliver Beutel

Covalent Monofunctionalization of Peptide-Coated Quantum Dots for Single-Molecule Assays

Fluorescent probes for biological imaging of single molecules (SM) have many stringent design requirements. In the case of quantum dot (QD) probes, it remains a challenge to control their functional properties with high precision. Here, we describe the simple preparation of QDs with reduced size and monovalency. Our approach combines a peptide surface coating, stable covalent conjugation of targeting units and purification by gel electrophoresis. We precisely characterize these probes by ensemble and SM techniques and apply them to tracking individual proteins in living cells.

Triple‐Color Super‐Resolution Imaging of Live Cells: Resolving Submicroscopic Receptor Organization in the Plasma Membrane

In living color: efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super-resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual-color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved.

Self-Controlled Monofunctionalization of Quantum Dots for Multiplexed Protein Tracking in Live Cells†

Tracking the motion of individual proteins on the surface of live cells has contributed considerably towards unveiling the functional organization of proteins in the plasma membrane. Individual proteins labeled with quantum dots (QDs) can be imaged over long time periods with ultrahigh spatial and temporal resolution, yielding powerful information on the spatiotemporal dynamics of proteins at the plasma membrane in live cells. A key challenge for the application of QDs is to site-specifically attach proteins to the surface of these nanoparticles in a stoichiometric manner without affecting protein function. Several procedures for rendering surfaces of QDs biocompatible have been described, thus reducing non-specific binding and protein denaturation on the QD surface. However, functionalized biocompatible QDs used to target cell surface proteins generally result in multipoint attachment to the target proteins because the number of functional groups on the QDs is very difficult to control. Such multiply functionalized QDs induce clustering of target proteins on the cell surface, biasing not only lateral diffusion but also the functional properties of these proteins. As a consequence, increased endocytosis has been observed upon binding of QDs functionalized with multiple epidermal grow factor (EGF) molecules to cell-surface EGF receptors. Stochastic functionalization of multiple reactive sites on the QD offers the choice of obtaining only a minor fraction of the QDs with a single functional group, or a significant fraction of QDs with multiple functional groups. Preparation of homogeneous, monofunctional QDs currently relies on electrophoretic purification, which has been achieved only for very small QDs. These compact QDs are designed with very thin surface coatings, which have the disadvantage of showing relatively strong non-specific interactions. Most approaches for targeting proteins using QDs in live cells are based on biotin–streptavidin interactions, which form quasi-irreversible complexes. For multiplexed, generic labeling of proteins on the cell surface, further targeting strategies are required. We have recently described tris(hydroxymethyl)methylamine–nitrilotriacetic acid (Tris-NTA) moieties for highly specific and stable attachment of fluorophores and other functional units to histidine-tagged proteins in vitro and on the surface of live cells. The lifetime of Tris-NTA complexes with His-tagged proteins is in the order of several hours, which is well-suited to medium-term single molecule tracking applications. Herein, we have attempted to control the functionalization degree of QDs with Tris-NTA by means of electrostatic repulsion. We devised a bottom-up coupling chemistry based on a novel Tris-NTA derivative (1; Figure 1a), which comprises a thiol-terminated hepta(ethylene glycol) linker. This compound was generated in situ by reduction of the disulfide-linked dimer (1a ; see the Supporting Information, Scheme S1) and coupled to commercially available polymer-coated and amine-functionalized QDs by means of a hetero-bifunctional cross-linker (Figure 1b). Covalently attachment of 1 to surfaces modified with maleimide-functionalized polyethylene glycol (PEG) polymer brush and specific immobilization of His-tagged proteins was confirmed by label-free detection (Supporting Information, Figure S1). To control the degree of functionalization with Tris-NTA on the QD surface, the reaction of 1 with surface maleimide groups was performed at low ionic strength. Under these conditions, all QDs were reacted with Tris-NTA, as confirmed by an increase in negative charges detected by anion exchange chromatography and agarose gel electrophoreses (Figure 1c,d). These assays indicated relatively monodisperse electrostatic properties after coupling of 1, despite the fact that it was reacted at a large excess (660 mm of compound 1 to 1 mm QD). Coupling of 1 at higher ionic strength yielded QDs with a substantially higher degree of functionalization, as confirmed by a further shift of the signals both in anion exchange chromatography and agarose gel electrophoresis (Figure 1c,d). To characterize the functional properties of Tris-NTAcoupled QDs, binding to immobilized hexahistidine (H6) [*] Dr. C. You, S. Wilmes, O. Beutel, S. L chte, Y. Podoplelowa, F. Roder, C. Richter, T. Seine, D. Schaible, Prof. Dr. J. Piehler Division of Biophysics, Universit t Osnabr ck Barbarstrasse 11, 49076 Osnabr ck (Germany) Fax: (+49)541-969-2262 E-mail: piehler@uos.de Homepage: http://www.biologie.uni-osnabrueck.de/Biophysik/ Piehler/

Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons

This is the first study in which the interaction of a microtubule-associated protein has been evaluated by direct single-molecule observations in living neurons. The data imply a novel kiss-and-hop mechanism of tau–microtubule interaction, rationalizing how tau can regulate microtubule dynamics without interfering with axonal transport.

Receptor dimerization dynamics as a regulatory valve for plasticity of type I interferon signaling

Quantitative single-molecule receptor dimerization assays show dimerization of IFNAR1 and IFNAR2 upon IFN treatment, and reveal the limiting role of IFNAR1 binding affinity in complex assembly and the regulatory role of USP18.

Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane

The use of micropatterned surfaces that bind HaloTag fusion proteins allows spatial organization of plasma membrane proteins for efficient visualization and quantification of protein–protein interactions in live cells.

Spatial organization of lipid phases in micropatterned polymer-supported membranes.

We have established an approach for the spatial control of lipid phase separation in tethered polymer-supported membranes (PSMs), which were obtained by vesicle fusion on a poly(ethylene glycol) polymer brush functionalized with fatty acid moieties. Phase separation of ternary lipid mixtures (1,2-dioleoyl-sn-glycero-3-phosphocholine/sphingomyelin/cholesterol) into liquid-disordered (l(d)) and liquid-ordered (l(o)) phases within both leaflets was obtained with palmitic acid as the anchoring group. In contrast, tethering of the PSM with oleic acid interfered with the phase separation in the surface-proximal leaflet. We exploited this feature for the assembly of l(o) domains within PSMs into defined structures by binary micropatterning of palmitic and oleic acid into complementary areas. Ternary lipid mixtures spontaneously separated into l(o) and l(d) phases controlled by the geometry of the underlying tethers. Transmembrane proteins reconstituted in these phase-separated PSMs strictly partitioned into the l(d) phase. Hence, the l(o) phase could be used for confining transmembrane proteins into microscopic and submicroscopic domains.

Maleimide Photolithography for Single-Molecule Protein-Protein Interaction Analysis in Micropatterns

Spatial organization of proteins into microscopic structures has important applications in fundamental and applied research. Preserving the function of proteins in such microstructures requires generic methods for site-specific capturing through affinity handles. Here, we present a versatile bottom-up surface micropatterning approach based on surface functionalization with maleimides, which selectively react with organic thiols. Upon UV irradiation through a photomask, the functionality of illuminated maleimide groups was efficiently destroyed. Remaining maleimides in nonilluminated regions were further reacted with different thiol-functionalized groups for site-specific protein immobilization under physiological conditions. Highly selective immobilization of His-tagged proteins into tris(nitrilotriacetic acid) functionalized microstructures with very high contrast was possible even by direct capturing of proteins from crude cell lysates. Moreover, we employed phosphopantetheinyl transfer from surface-immobilized coenzyme A to ybbR-tagged proteins in order to implement site-specific, covalent protein immobilization into microstructures. The functional integrity of the immobilized protein was confirmed by monitoring protein-protein interactions in real time. Moreover, we demonstrate quantitative single-molecule analysis of protein-protein interactions with proteins selectively captured into these high-contrast micropatterns.

Electrostatically controlled quantum dot monofunctionalization for interrogating the dynamics of protein complexes in living cells.

Quantum dots (QD) are powerful labels for probing diffusion and interaction dynamics of proteins on the single molecule level in living cells. Protein cross-linking due to multifunctional QD strongly affects these properties. This becomes particularly critical when labeling interaction partners with QDs for interrogating the dynamics of complexes. We have here implemented a generic method for QD monofunctionalization based on electrostatic repulsion of a highly negatively charged peptide carrier. On the basis of this method, monobiotinylated QDs were prepared with high yield as confirmed by single molecule assays. These QDs were successfully employed for probing the assembly and diffusion dynamics of binary and ternary cytokine-receptor complexes on the surface of living cells by dual color single QD tracking. Thus, sequential and dynamic recruitment of the type I interferon receptor subunits by the ligand could be observed.

High‐Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells

Lipid analogues carrying three nitrilotriacetic acid (tris-NTA) head groups were developed for the selective targeting of His-tagged proteins into liquid ordered (lo ) or liquid disordered (ld ) lipid phases. Strong partitioning into the lo phase of His-tagged proteins bound to tris-NTA conjugated to saturated alkyl chains (tris-NTA DODA) was achieved, while tris-NTA conjugated to an unsaturated alkyl chain (tris-NTA SOA) predominantly resided in the ld phase. Interestingly, His-tag-mediated lipid crosslinking turned out to be required for efficient targeting into the lo phase by tris-NTA DODA. Robust partitioning into lo phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into lo and ld domains within the plasma membrane of living cells was demonstrated by single-molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.