7 research outputs found
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 polyÂ(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 ATPâprotein 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 Ă
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>, 109â113), 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 NH<sub>2</sub> groups. In the past, various tricks or detours were applied to minimize the undesired bivalent reaction of bifunctional linkers with adjacent NH<sub>2</sub> 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 (âacetal-PEG-NHSâ) 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, 1â10 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 (âaldehyde-PEG-NHSâ) to adjacent NH<sub>2</sub> 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