Changjiang You

High-Affinity Labeling and Tracking of Individual Histidine-Tagged Proteins in Live Cells Using Ni2+ Tris-nitrilotriacetic Acid Quantum Dot Conjugates

Investigation of many cellular processes using fluorescent quantum dots (QDs) is hindered by the nontrivial requirements for QD surface functionalization and targeting. To address these challenges, we designed, characterized and applied QD-trisNTA, which integrates tris-nitrilotriacetic acid, a small and high-affinity recognition unit for the ubiquitous polyhistidine protein tag. Using QD-trisNTA, we demonstrate two-color QD tracking of the type-1 interferon receptor subunits in live cells, potentially enabling direct visualization of protein-protein interactions at the single molecule level.

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.

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/

Affinity capturing for targeting proteins into micro and nanostructures

Protein immobilization into micro and nanoscaled patterns opens exciting possibilities in fundamental and applied research. Developing efficient capturing techniques while preserving the structural and functional integrity of the proteins on surfaces is a key challenge for surface scientists. In this paper, current techniques for site-specific protein immobilization into engineered surface architectures are reviewed. Fundamental principles for functional protein immobilization on solid supports are discussed and popular affinity-based recognition pairs and their application for capturing proteins into nano and microstructures are presented.

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.

Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling

Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD–LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.

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.

TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1’s 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼9–17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼2.8–3.6 κBT greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This ‘tag-team proofreading’ represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.

Selective Targeting of Fluorescent Nanoparticles to Proteins Inside Live Cells

Single-molecule localization and tracking techniques have contributed towards observing the spatiotemporal organization of proteins in the plasma membrane. Individual proteins labeled with fluorescent nanoparticles (FNPs) can be imaged over long time with ultrahigh spatial and temporal resolution. A key challenge for the biophysical application of FNPs, however, is to site-specifically target the nanoparticles to proteins in living cells. For selective labeling of cell surface proteins with FNPs, biomolecules such as antibodies and streptavidin have been employed as well as chemical recognition based on immobilized transition-metal ions. These recognition units, however, are not compatible with FNP targeting to proteins inside living cells, because the structural integrity of antibodies is often affected by the reducing conditions in the cytoplasm, streptavidin is blocked with endogenous biotin, and transition-metal ions are coordinated by cysteine-rich proteins. Intracellular FNP targeting is furthermore challenging, because blocking of nonspecific binding and washing out of nonbound FNPs is not possible in intact cells. Here, we aimed to establish a highly specific and efficient approach for FNP targeting inside live cells, which overcomes these particular challenges. As a biochemical recognition system compatible with the cytoplasm, we employed an enzymatic covalent labeling approach based on the HaloTag. This engineered dehalogenase irreversibly reacts with a chlorohexane moiety (HaloTag ligand, HTL) attached to fluorescent dyes and other probes. This highly specific reaction has been exploited for protein labeling in live cells. We attempted functionalization of FNPs with HTL through maleimide/thiol-chemistry as well as by using different biotin derivatives. However, neither significant specific binding to the immobilized HaloTag was observed in vitro, nor efficient targeting upon microinjection into live cells expressing HaloTag fusion proteins using a microcapillary. For this reason, we characterized the association kinetics of different derivatives of the HTL in more detail using realtime surface-sensitive detection by simultaneous reflectance interference (RIF) and total internal reflection fluorescence spectroscopy (TIRFS) detection. For this purpose, purified HaloTag with a His-tag (His= histidine, HaloTag-H12) was site-specifically immobilized on a polyethylene glycol (PEG) polymer brush functionalized with tris(nitrilotriacetic acid), tris-NTA, and binding of fluorescent substrates was monitored in real time (see Figure S1 in the Supporting Information). Rapid binding of HTL conjugated with the fluorescent dye AlexaFluor 488 (HTL) was detected by TIRFS (see Figure S1 in the Supporting Information), yielding a reaction rate constant of 1 10m 1 s . To directly compare the reaction rate constants of fluorescent and nonfluorescent HTL conjugates (see Scheme S1 in the Supporting Information), a competition assay was established with HTL as a fluorescent tracer (see Figure S2 in the Supporting Information). These rate constants are summarized in Table S1 in the Supporting Information. Strikingly, a substantially faster reaction rate constant of 1 10m 1 s 1 was observed for HTL conjugated with tetramethylrhodamine (HTL), which is similar to the published rate constant of this reaction measured in solution (2.7 10m 1 s ). Surprisingly, an elongated ethylene glycol linker substantially reduced the reaction rate constant. Even slower rate constants were obtained for biotinylated and for unmodified HTL (around 10m 1 s ). These results suggested that the conjugated fluorescence dyes play a critical role for the association kinetics, probably by stabilizing the noncovalent enzyme–substrate complex, by hydrophobic interactions, prior to the ester formation by reaction with D106 in the binding pocket of the HaloTag. Strikingly, engineering of the HaloTag from the original dehalogenase involved incorporation of hydrophobic residues in the proximity of the reactive site. Based on the observation that hydrophobic as well as positively charged residues increase the reaction rate constant of HTL derivatives, we implemented a novel approach for surface functionalization with the HTL based on click chemistry using commercially available dibenzocyclooctynelike (DBCO) derivatives and azide-functionalized HTL (Figure 1a and Scheme S2 in the Supporting Information). Thus a hydrophobic moiety was integrated to HTL similar to the HTL-dye conjugates. The reaction kinetics of the HTL derivative obtained by this reaction (clickHTL) was compared with other HTL-derivatives of the competitive binding [*] D. Lise, Dr. C. You, Prof. Dr. J. Piehler Division of Biophysics, Department of Biology Universit t Osnabr ck, Barbarastrasse 11 49076 Osnabr ck (Germany) E-mail: piehler@uos.de Homepage: http://www.biologie.uni-osnabrueck.de/Biophysik/ Piehler/

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.