4 research outputs found
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Ion-Specific Control of the Self-Assembly Dynamics of a Nanostructured Protein Lattice
Self-assembling proteins offer a potential means of creating nanostructures with complex structure and function. However, using self-assembly to create nanostructures with long-range order whose size is tunable is challenging, because the kinetics and thermodynamics of protein interactions depend sensitively on solution conditions. Here we systematically investigate the impact of varying solution conditions on the self-assembly of SbpA, a surface-layer protein from <i>Lysinibacillus sphaericus</i> that forms two-dimensional nanosheets. Using high-throughput light scattering measurements, we mapped out diagrams that reveal the relative yield of self-assembly of nanosheets over a wide range of concentrations of SbpA and Ca<sup>2+</sup>. These diagrams revealed a localized region of optimum yield of nanosheets at intermediate Ca<sup>2+</sup> concentration. Replacement of Mg<sup>2+</sup> or Ba<sup>2+</sup> for Ca<sup>2+</sup> indicates that Ca<sup>2+</sup> acts both as a specific ion that is required to induce self-assembly and as a general divalent cation. In addition, we use competitive titration experiments to find that 5 Ca<sup>2+</sup> bind to SbpA with an affinity of 67.1 ± 0.3 μM. Finally, we show <i>via</i> modeling that nanosheet assembly occurs by growth from a negligibly small critical nucleus. We also chart the dynamics of nanosheet size over a variety of conditions. Our results demonstrate control of the dynamics and size of the self-assembly of a nanostructured lattice, the constituents of which are one of a class of building blocks able to form novel hybrid nanomaterials
Ion-Specific Control of the Self-Assembly Dynamics of a Nanostructured Protein Lattice
Self-assembling proteins offer a potential means of creating nanostructures with complex structure and function. However, using self-assembly to create nanostructures with long-range order whose size is tunable is challenging, because the kinetics and thermodynamics of protein interactions depend sensitively on solution conditions. Here we systematically investigate the impact of varying solution conditions on the self-assembly of SbpA, a surface-layer protein from <i>Lysinibacillus sphaericus</i> that forms two-dimensional nanosheets. Using high-throughput light scattering measurements, we mapped out diagrams that reveal the relative yield of self-assembly of nanosheets over a wide range of concentrations of SbpA and Ca<sup>2+</sup>. These diagrams revealed a localized region of optimum yield of nanosheets at intermediate Ca<sup>2+</sup> concentration. Replacement of Mg<sup>2+</sup> or Ba<sup>2+</sup> for Ca<sup>2+</sup> indicates that Ca<sup>2+</sup> acts both as a specific ion that is required to induce self-assembly and as a general divalent cation. In addition, we use competitive titration experiments to find that 5 Ca<sup>2+</sup> bind to SbpA with an affinity of 67.1 ± 0.3 μM. Finally, we show <i>via</i> modeling that nanosheet assembly occurs by growth from a negligibly small critical nucleus. We also chart the dynamics of nanosheet size over a variety of conditions. Our results demonstrate control of the dynamics and size of the self-assembly of a nanostructured lattice, the constituents of which are one of a class of building blocks able to form novel hybrid nanomaterials
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Glycosylated Peptoid Nanosheets as a Multivalent Scaffold for Protein Recognition
Glycoproteins adhered on the cellular
membrane play a pivotal role
in a wide range of cellular functions. Their importance is particularly
relevant in the recognition process between infectious pathogens (such
as viruses, bacteria, toxins) and their host cells. Multivalent interactions
at the pathogen-cell interfaces govern binding events and can result
in a strong and specific interaction. Here we report an approach to
mimic the cell surface presentation of carbohydrate ligands by the
multivalent display of sugars on the surface of peptoid nanosheets.
The constructs provide a highly organized 2D platform for recognition
of carbohydrate-binding proteins. The sugars were displayed using
different linker lengths or within loops containing 2–6 hydrophilic
peptoid monomers. Both the linkers and the loops contained one alkyne-bearing
monomer, to which different saccharides were attached by copper-catalyzed
azide–alkyne cycloaddition reactions. Peptoid nanosheets functionalized
with different saccharide groups were able to selectively bind multivalent
lectins, Concanavalin A and Wheat Germ Agglutinin, as observed by
fluorescence microscopy and a homogeneous Förster resonance
energy transfer (FRET)-based binding assay. To evaluate the potential
of this system as sensor for threat agents, the ability of functionalized
peptoid nanosheets to bind Shiga toxin was also studied. Peptoid nanosheets
were functionalized with globotriose, the natural ligand of Shiga
toxin, and the effective binding of the nanomaterial was verified
by the FRET-based binding assay. In all cases, evidence for multivalent
binding was observed by systematic variation of the ligand display
density on the nanosheet surface. These cell surface mimetic nanomaterials
may find utility in the inactivation of pathogens or as selective
molecular recognition elements
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The Molecular Basis for Binding of an Electron Transfer Protein to a Metal Oxide Surface
Achieving
fast electron transfer between a material and protein
is a long-standing challenge confronting applications in bioelectronics,
bioelectrocatalysis, and optobioelectronics. Interestingly, naturally
occurring extracellular electron transfer proteins bind to and reduce
metal oxides fast enough to enable cell growth, and thus could offer
insight into solving this coupling problem. While structures of several
extracellular electron transfer proteins are known, an understanding
of how these proteins bind to their metal oxide substrates has remained
elusive because this abiotic–biotic interface is inaccessible
to traditional structural methods. Here, we use advanced footprinting
techniques to investigate binding between the <i>Shewanella oneidensis</i> MR-1 extracellular electron transfer protein MtrF and one of its
substrates, α-Fe<sub>2</sub>O<sub>3</sub> nanoparticles, at
the molecular level. We find that MtrF binds α-Fe<sub>2</sub>O<sub>3</sub> specifically, but not tightly. Nanoparticle binding
does not induce significant conformational changes in MtrF, but instead
protects specific residues on the face of MtrF likely to be involved
in electron transfer. Surprisingly, these residues are separated in
primary sequence, but cluster into a small 3D putative binding site.
This binding site is located near a local pocket of positive charge
that is complementary to the negatively charged α-Fe<sub>2</sub>O<sub>3</sub> surface, and mutational analysis indicates that electrostatic
interactions in this 3D pocket modulate MtrF–nanoparticle binding.
Strikingly, these results show that binding of MtrF to α-Fe<sub>2</sub>O<sub>3</sub> follows a strategy to connect proteins to materials
that resembles the binding between donor–acceptor electron
transfer proteins. Thus, by developing a new methodology to probe
protein–nanoparticle binding at the molecular level, this work
reveals one of nature’s strategies for achieving fast, efficient
electron transfer between proteins and materials