We aspired to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, as tools for selectively attaching spectroscopic probes and other functional elements to recombinant proteins. Several supramolecular entities containing 2-4 nitrilotriacetic acid (NTA) moieties were synthesized, which additionally contained an amino group, to which fluorescein was coupled as a sensitive reporter probe. These multivalent chelator heads (MCH) (termed bis-, tris-, and tetrakis-NTA) were characterized with respect to their interaction with hexahistidine (H6)- and decahistidine (H10)-tagged targets. Substantially increased binding stability with increasing number of NTA moieties was observed by analytical size exclusion chromatography. The binding enthalpies as determined by isothermal titration calorimetry increased nearly additively with the number of possible coordinative bonds between chelator heads and tags. Yet, a substantial excess of histidines in the oligohistidine tag was required for obtaining fully additive binding enthalpies. Dissociation kinetics of MCH/oligohistidine complexes measured by fluorescence dequenching showed an increase in stability by 4 orders of magnitude compared to that of mono-NTA, and subnanomolar affinity was reached for tris-NTA. The gain in free energy with increasing multivalency was accompanied by an increasing loss of entropy, which was ascribed to the high flexibility of the binding partners. Numerous applications of these MCHs for noncovalent, high affinity, yet reversible tethering of spectroscopic probes and other functional elements to the recombinant proteins can be envisioned.

During intracellular membrane trafficking and remodeling, protein complexes known as the ESCRTs interact with membranes and are required for budding processes directed away from the cytosol, including the budding of intralumenal vesicles to form multivesicular bodies, for the budding of some enveloped viruses, and for daughter cell scission in cytokinesis. Here we found that the ESCRT-III proteins CHMP2A and CHMP3 could assemble in vitro into helical tubular structures that expose their membrane interaction sites on the outside of the tubule while the AAA-type ATPase VPS4 could bind on the inside of the tubule and disassemble the tubes upon ATP hydrolysis. CHMP2A and CHMP3 co-polymerized in solution and their membrane targeting was cooperatively enhanced on planar lipid bilayers. Such helical CHMP structures could thus assemble within the neck of an inwardly-budding vesicle, catalyzing late steps in budding under the control of VPS4.

Labeling of proteins with fluorescent dyes offers powerful means for monitoring protein interactions in vitro and in live cells. Only a few techniques for noncovalent fluorescence labeling with well-defined localization of the attached dye are currently available. Here, we present an efficient method for site-specific and stable noncovalent fluorescence labeling of histidine-tagged proteins. Different fluorophores were conjugated to a chemical recognition unit bearing three NTA moieties (tris-NTA). In contrast to the transient binding of conventional mono-NTA, the multivalent interaction of tris-NTA conjugated fluorophores with oligohistidine-tagged proteins resulted in complex lifetimes of more than an hour. The high selectivity of tris-NTA toward cumulated histidines enabled selective labeling of proteins in cell lysates and on the surface of live cells. Fluorescence labeling by tris-NTA conjugates was applied for the analysis of a ternary protein complex in solution and on surfaces. Formation of the complex and its stoichiometry was studied by analytical size exclusion chromatography and fluorescence quenching. The individual interactions were dissected on solid supports by using simultaneous mass-sensitive and multicolor fluorescence detection. Using these techniques, formation of a 1:1:1 stoichiometry by independent interactions of the receptor subunits with the ligand was shown. The incorporation of transition metal ions into the labeled proteins upon labeling with tris-NTA fluorophore conjugates provided an additional sensitive spectroscopic reporter for detecting and monitoring protein-protein interactions in real time. A broad application of these fluorescence conjugates for protein interaction analysis can be envisaged.

ESCRT-III proteins catalyze membrane fission during multi vesicular body biogenesis, budding of some enveloped viruses and cell division. We suggest and analyze a novel mechanism of membrane fission by the mammalian ESCRT-III subunits CHMP2 and CHMP3. We propose that the CHMP2-CHMP3 complexes self-assemble into hemi-spherical dome-like structures within the necks of the initial membrane buds generated by CHMP4 filaments. The dome formation is accompanied by the membrane attachment to the dome surface, which drives narrowing of the membrane neck and accumulation of the elastic stresses leading, ultimately, to the neck fission. Based on the bending elastic model of lipid bilayers, we determine the degree of the membrane attachment to the dome enabling the neck fission and compute the required values of the protein-membrane binding energy. We estimate the feasible values of this energy and predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission. We support the computational model by electron tomography imaging of CHMP2-CHMP3 assemblies in vitro. We predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission.

We present a generic approach for immobilizing oligohistidine-tagged proteins with high stability and homogeneous functionality onto glass-type surfaces. Multivalent chelator heads (MCH) carrying two and three nitrilotriacetic acid (NTA) moieties were coupled with controlled surface concentration to glass surfaces premodified with an ultrathin two-dimensional polymer brush of a bifunctional poly(ethylene glycol). Low roughness and lateral homogeneity of these surfaces were confirmed by AFM and fluorescence microscopy, respectively. Protein immobilization and interactions at these interfaces were studied by label-free and fluorescence detection. Oligohistidine-tagged proteins bound specifically to NTA loaded with nickel(II) ions and could be eluted with imidazole. More than 90% of the immobilized protein preserved its activity. In contrast to mono-NTA, immobilized multivalent chelator heads bound oligohistidine-tagged proteins stoichiometrically and with high stability, even at very low chelator surface concentrations. Thus, an excess of the metal chelator sites was not necessary, and excessive binding sites could be quantitatively blocked with an indifferent protein. As a consequence, increased functional stability of the immobilized protein and a substantial reduction in nonspecific adsorption were achieved. Binding of histidine-tagged proteins to the MCH-modified surface was efficiently blocked by stoichiometric amounts of soluble MCH, and biomolecular interaction unbiased by the interaction of the histidine tag to the surface-bound MCH was observed. These excellent features and the compatibility with many solid-phase analytical techniques make this surface chemistry beneficial for functional protein analysis.

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ALIX recruits ESCRT-III CHMP4 and is involved in membrane remodeling during endosomal receptor sorting, budding of some enveloped viruses and cytokinesis. We show that ALIX dimerizes via the middle domain (ALIX-V) in solution. Structural modeling based on small angle X-ray scattering (SAXS) data reveal an elongated crescent shaped conformation for dimeric ALIX lacking the proline rich domain (ALIXBRO1-V). Mutations at the dimerization interface prevent dimerization and induce an open elongated monomeric conformation of ALIX-V as determined by SAXS modeling. ALIX dimerizes in vivo and dimeric ALIX co-localizes with CHMP4B upon co-expression. We show further that ALIX dimerization affects HIV-1 budding. C-terminally truncated activated CHMP4B retaining the ALIX binding site forms linear, circular and helical filaments in vitro, which can be bridged by ALIX. Our data suggest that dimeric ALIX represents the active form that interacts with ESCRT-III CHMP4 polymers and functions as a scaffolding protein during membrane remodeling processes.

Protein micro-/nanoarrays are becoming increasingly important in systematic approaches for the exploration of protein-protein interactions and dynamic protein networks, so there is a high demand for specific, generic, stable, uniform, and locally addressable protein immobilization on solid supports. Here we present multivalent metal-chelating thiols that are suitable for stable binding of histidine-tagged proteins on biocompatible self-assembled monolayers (SAMs). The architectures and physicochemical properties of these SAMs have been probed by various surface-sensitive techniques such as contact angle goniometry, ellipsometry, and infrared reflection-absorption spectroscopy. The specific molecular organization of proteins and protein complexes was demonstrated by surface plasmon resonance, confocal laser scanning, and atomic force microscopy. In contrast to the mono-NTA/His6 tag interaction, which has major drawbacks because of its low affinity and fast dissociation, drastically improved stability of protein binding by these multivalent chelator surfaces was observed. The immobilized histidine-tagged proteins are uniformly oriented and retain their function. At the same time, proteins can be removed from the chip surface under mild conditions (switchability). This new platform for switchable and oriented immobilization should assist proteome-wide wide analyses of protein-protein interactions as well as structural and single-molecule studies.