Painting with Biomolecules at the Nanoscale: Biofunctionalization with Tunable Surface Densities

We present a generic and flexible method to nanopattern biomolecules on surfaces. Carbon-containing nanofeatures are written at variable diameter and spacing by a focused electron beam on a poly­(ethylene glycol) (PEG)-coated glass substrate. Proteins physisorb to the nanofeatures with remarkably high contrast factors of more than 1000 compared to the surrounding PEG surfaces. The biological activity of model proteins can be retained as shown by decorating avidin spots with biotinylated DNA, thereby underscoring the universality of the nano-biofunctionalized platform for the binding of other biotinylated ligands. In addition, biomolecule densities can be tuned over several orders of magnitude within the same array, as demonstrated by painting a microscale image with nanoscale pixels. We expect that these unique advantages open up entirely new ways to design biophysical experiments, for instance, on cells that respond to the nanoscale densities of activating molecules

High-Speed AFM Images of Thermal Motion Provide Stiffness Map of Interfacial Membrane Protein Moieties

The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges

High-Speed AFM Images of Thermal Motion Provide Stiffness Map of Interfacial Membrane Protein Moieties

The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges

High-Speed AFM Images of Thermal Motion Provide Stiffness Map of Interfacial Membrane Protein Moieties

The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges

Mapping the Nucleotide Binding Site of Uncoupling Protein 1 Using Atomic Force Microscopy

A tight regulation of proton transport in the inner mitochondrial membrane is crucial for physiological processes such as ATP synthesis, heat production, or regulation of the reactive oxygen species as proposed for the uncoupling protein family members (UCP). Specific regulation of proton transport is thus becoming increasingly important in the therapy of obesity and inflammatory, neurodegenerative, and ischemic diseases. We and other research groups have shown previously that UCP1- and UCP2-mediated proton transport is inhibited by purine nucleotides. Several hypotheses have been proposed to explain the inhibitory effect of ATP, although structural details are still lacking. Moreover, the unresolved mystery is how UCP operates in vivo despite the permanent presence of high (millimolar) concentrations of ATP in mitochondria. Here we use the topographic and recognition (TREC) mode of an atomic force microscope to visualize UCP1 reconstituted into lipid bilayers and to analyze the ATP–protein interaction at a single molecule level. The comparison of recognition patterns obtained with anti-UCP1 antibody and ATP led to the conclusion that the ATP binding site can be accessed from both sides of the membrane. Using cantilever tips with different cross-linker lengths, we determined the location of the nucleotide binding site inside the membrane with 1 Å precision. Together with the recently published NMR structure of a UCP family member (Berardi et al. <i>Nature</i>, <b>2011</b>, <i>476</i>, 109–113), our data provide a valuable insight into the mechanism of the nucleotide binding and pave the way for new pharmacological approaches against the diseases mentioned above

Single Molecular Recognition Force Spectroscopy Study of a Luteinizing Hormone-Releasing Hormone Analogue as a Carcinoma Target Drug

The luteinizing hormone-releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40), is a candidate target drug associated with elevated LHRH receptor (LHRH-R) expression in malignant tumor tissue. The capability of LHRH-PE40 to recognize LHRH-Rs on a living cell membrane was studied with single molecular recognition force spectroscopy (SMFS) based on atomic force microscopy (AFM). The recognition force of LHRH-PE40/LHRH-R was compared with that of LHRH/LHRH-R by dynamic force spectroscopy. Meanwhile, cell growth inhibition assay and fluorescence imaging were presented as complementary characterization. The results show that LHRH moiety keeps its capability to recognize LHRH-R specifically, which implies that recombinant protein LHRH-PE40 can be a promising target drug

Linking of Sensor Molecules with Amino Groups to Amino-Functionalized AFM Tips

The measuring tip of an atomic force microscope (AFM) can be upgraded to a specific biosensor by attaching one or a few biomolecules to the apex of the tip. The biofunctionalized tip is then used to map cognate target molecules on a sample surface or to study biophysical parameters of interaction with the target molecules. The functionality of tip-bound sensor molecules is greatly enhanced if they are linked via a thin, flexible polymer chain. In a typical scheme of tip functionalization, reactive groups are first generated on the tip surface, a bifunctional cross-linker is then attached with one of its two reactive ends, and finally the probe molecule of interest is coupled to the free end of the cross-linker. Unfortunately, the most popular functional group generated on the tip surface is the amino group, while at the same time, the only useful coupling functions of many biomolecules (such as antibodies) are also NH₂ groups. In the past, various tricks or detours were applied to minimize the undesired bivalent reaction of bifunctional linkers with adjacent NH₂ groups on the tip surface. In the present study, an uncompromising solution to this problem was found with the help of a new cross-linker (“acetal-PEG-NHS”) which possesses one activated carboxyl group and one acetal-protected benzaldehyde function. The activated carboxyl ensures rapid unilateral attachment to the amino-functionalized tip, and only then is the terminal acetal group converted into the amino-reactive benzaldehyde function by mild treatment (1% citric acid, 1–10 min) which does not harm the AFM tip. As an exception, AFM tips with magnetic coating become demagnetized in 1% citric acid. This problem was solved by deprotecting the acetal group before coupling the PEG linker to the AFM tip. Bivalent binding of the corresponding linker (“aldehyde-PEG-NHS”) to adjacent NH₂ groups on the tip was largely suppressed by high linker concentrations. In this way, magnetic AFM tips could be functionalized with an ethylene diamine derivative of ATP which showed specific interaction with mitochondrial uncoupling protein 1 (UCP1) that had been purified and reconstituted in a mica-supported planar lipid bilayer