[show abstract][hide abstract] ABSTRACT: There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle ((AuNP)tris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of (AuNP)tris-NTA labeled proteins by electron microscopy is further ensured by the reagent's short conformationally restricted linker. We used (AuNP)tris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. (AuNP)tris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our labeling reagent should find broad application in noncovalent, site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies.
[show abstract][hide abstract] ABSTRACT: Local interactions between the tips of microtubules and the cell cortex, or other cellular components such as kinetochores, play an important role in essential cellular processes like establishing cell polarity, distribution of organelles, and microtubule aster and chromosome positioning. Here we present two in vitro assays that specifically mimic microtubule-cortex interactions by employing selectively functionalized microfabricated barriers that allow for the immobilization of proteins with a range of affinities. We describe the microfabrication process to create gold or glass barriers and the subsequent functionalization of these barriers using self-assembled thiol monolayers or polylysine-poly(ethylene glycol), respectively. Near-permanent attachment of proteins is obtained using biotinylated surfaces combined with streptavidin and biotinylated proteins. Lower affinity interactions, further tunable with the addition of imidazole, are obtained using nickel-nitrilotriacetic acid (Ni(II)-NTA) functionalization combined with his-tagged proteins. Both mono-NTA and tris-NTA compounds are used. We show an assay to reconstitute the "end-on" interaction between dynamic microtubule tips and barrier-attached dynein, mimicking the cellular situation at the cortex and at kinetochores. In a second assay, we reconstitute microtubule-based delivery of end-tracking proteins to functionalized barriers, mimicking the transport of cell-end markers to the cell poles in interphase fission yeast cells.
Methods in cell biology 01/2014; 120C:69-90. · 1.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Angewandte Chemie International Edition 12/2013; · 13.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Nucleic Acids Research 11/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Localized surface plasmon resonance (LSPR) offers powerful means for sensitive label-free detection of protein-protein interactions in a highly multiplexed format. We have here established self-assembly and surface modification of plasmonic nanostructures on solid support suitable for quantitative protein-protein interaction analysis by spectroscopic and microscopic LSPR detection. These architectures were obtained by layer-by-layer assembly via electrostatic attraction. Gold nanoparticles (AuNP) were adsorbed on a biocompatible amine-terminated polyethylene glycol (PEG) polymer brush and further functionalized by poly-L-lysine graft PEG (PLL-PEG) copolymers. Stable, yet reversible protein immobilization was achieved via tris-(nitrilotriacetic acid) groups incorporated into the PLL-PEG coating. Thus, site-specific immobilization of His-tagged proteins via complexed Ni(II) ions was achieved. Functional protein immobilization on the surface was confirmed by real-time detection of LSPR scattering by reflectance spectroscopy. Association and dissociation rate constants obtained for a reversible protein-protein interaction were in good agreement with the data obtained by other surface-sensitive detection techniques. For spatially-resolved detection, AuNP were assembled into micropatterns by means of photolithographic uncaging of surface amines. LSPR imaging of reversible protein-protein interactions was possible in a conventional wide field microscope yielding detection limits of ~30 protein molecules within a diffraction limited surface area.
[show abstract][hide abstract] ABSTRACT: TFIIF-a general transcription factor comprising two subunits Tfg1 and Tfg2, can tightly associate with RNA polymerase II (RNAPII) to regulate the mRNA synthesis in yeast. Herein, to localize the C-terminus of Tfg2 in the RNAPII-TFIIF architecture, we devise a two-stage hybrid approach using electron microscopy (EM) and Förster resonance energy transfer (FRET). In the first stage, we label the poly-histidine appended to the Tfg2 C-terminus with nickel-NTA nanogold and use a seven-step single particle image analysis protocol to limit the nanogold-accessible region next to the RNAPII clamp. In the second stage, for, Rpb2 and Rpb4 subunits of RNAPII proximal to the clamp are chosen for placing FRET satellites to enable nano-positioning (NP) analysis for improving the precision of localization. NP analysis shows the Tfg2 C-terminus is localized on the ridge of the clamp but may move to the top during transcription while the template DNA could be reciprocally perturbed by TFIIF.In summary, our EM-FRET hybrid method has mapped the Tfg2 C-terminus in the RNAPII-TFIIF and henceforth represents a novel and effective approach for elucidating the protein complex to nanometer precision.
Journal of Structural Biology 05/2013; · 3.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 interactions 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. Based on this method, mono-biotinylated 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.
ACS Chemical Biology 11/2012; · 5.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Analytical and Bioanalytical Chemistry 02/2009; 393(6-7):1563-70. · 3.66 Impact Factor