Macromolecular crowding has long been known to significantly affect protein oligomerization, and yet no direct quantitative measurements appear to have been made of its effects on the binding free energy of the elemental step of adding a single subunit. Here, we report the effects of two crowding agents on the binding free energy of two subunits in the Escherichia coli polymerase III holoenzyme. The crowding agents are found, paradoxically, to have only a modest stabilizing effect, of the order of 1 kcal/mol, on the binding of the two subunits. Systematic variations in the level of stabilization with crowder size are nevertheless observed. The data are consistent with theoretical predictions based on atomistic modeling of excluded-volume interactions with crowders. We reconcile the apparent paradox presented by our data by noting that the modest effects of crowding on elemental binding steps are cumulative, and thus lead to substantial stabilization of higher oligomers. Correspondingly, the effects of small variations in the level of crowding during the lifetime of a cell may be magnified, suggesting that crowding may play a role in increased susceptibility to protein aggregation-related diseases with aging.

The crowded environments inside cells can have significant effects on the folding stability and other biophysical properties of proteins. In this study on how macromolecular crowding affects protein folding, we took a significant step toward realistically mimicking intracellular environments by using a mixture of two crowding agents, Ficoll and dextran. We found that the mixed crowding exerts a greater stabilizing effect than the sum of the two individual crowding agents. Therefore, the composition of crowders, not just the total concentration, has a significant influence on the effects of crowding on protein folding. Since the composition of intracellular macromolecules varies within the lifetime of a cell, our finding may provide an explanation for age being an important risk factor for protein aggregation-related diseases such as Alzheimer’s disease and Parkinson’s disease.

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Background: Functional collagen fibrils are formed through the self-assembly of collagen triple helices. Results: A designed triple helical peptide self-assembles into collagen-like mini-fibrils. Conclusion:The sequence of the triple helix alone is sufficient to "code" for the axial, periodic structure of the mini-fibrils. Significance: The work demonstrates an approach to achieve collagen-like fibrils through the self-assembly of designed triple helices.

Selective removal of aqueous mercury to levels below 10 ng L−1 or part per trillion remains an elusive goal for public health and environmental agencies. Here, it is shown that a low‐cost nanocomposite sponge prepared by growing selenium (Se) nanomaterials on the surface and throughout the bulk of a polyurethane sponge exhibits a record breaking‐mercury ion (Hg2+) removal rate, regardless of the pH. The exposure of aqueous solutions containing 10 mg L−1–12 ng L−1 Hg2+ to the sponge for a few seconds results in clean water with undetectable mercury levels (detection limit: 0.2 ng L−1). Such performance is far below the acceptable limits in drinking water (2 µg L−1), industrial effluents (0.2 µg L−1), and the most stringent surface water quality standards (1.3 ng L−1). The sponge shows a unique preference for Hg, does not retain water nutrients, and can significantly reduce the concentration of other heavy metal pollutants. Furthermore, the sponge shows no cytotoxic effect on human cells while exhibiting strong antimicrobial properties. The high affinity of Hg for Se results in irreversible sequestration and detoxification of mercury by the sponge, confirming the suitability for landfill disposal.

Recently, various metal and semiconductor nanowires have been developed as building blocks for electronics, optics, and sensors. Among these newly developed nanowires, nanowires grown on biomolecular templates such as DNA and peptide assemblies are advantageous since the molecular recognition functions of these biomolecules with specific ligands can be employed to immobilize nanowires onto specific locations to establish desired device geometries. [1][2][3] However, most of the biomolecular-nanowire templates made from DNAs or peptides do not possess suitable electric properties for those devices, and therefore there is an extensive effort in the field of bionanotechnology to coat these addressable biomolecular nanowires with metals and semiconductors. [4][5][6][7][8][9][10][11][12][13][14][15][16] Recently, the morphology of coating on these peptide-nanotube templates was shown to be controlled by means of changing the peptide sequences and conformations, thus fine-tuning the electronic structures of resulting nanowires for their device applications. [17][18][19] While these biomolecular-nanowire templates appear to be promising building blocks for nanodevices, it is essential to have size monodispersity, strength, and mass producibility to impact real-world applications. For example, biomolecular templates self-assembled from peptidic monomers tend to yield polydisperse materials with heterogeneous diameters and uncontrolled length through the self-assembly process. The tobacco mosaic virus (TMV), a rod-shaped biomolecular template, has been applied for various metal coatings, however accurate control of the length with low dispersity is not an easy task. [20,21] The other type-DNA biomolecular templates-have defined lengths determined by the number of nucleic acids, however they lack conformational rigidity. The tendency of supertwisting of the double-helix DNA structure makes it difficult to obtain rigid and straight nanowires. Their production cost and time may also not be suitable for large-scale syntheses.Herein we report a new application using a collagen-like triple helix as a template nanowire which appears to overcome some of the shortcomings of other biomolecular templates. The collagen-like triple helix is the genetically engineered polypeptide assembly that contains a fragment from the natural collagen sequence. Our study demonstrates that by using the recombinant technology, we can design and amplify a collagen-like triple helix that is monodisperse, easily mineralized with metal ions, and can, thus, be applied as rigid biomolecular templates for metal-nanowire fabrications. Collagens are the major components of extracellular matrices for bones, cartilages, skins, blood vessels, and corneas, and they are the most abundant proteins in higher organisms with superior mechanical properties. [22][23][24] The collagen-like triple helix is made of three polypeptide chains tightly twisted and bundled together to form a rigid, rod-shaped molecule that is suitable for applications in building blocks of nanod...

The clinical severity of Osteogenesis Imperfecta (OI), also known as the brittle bone disease, relates to the extent of conformational changes in the collagen triple helix induced by Gly substitution mutations. The lingering question is why Gly substitutions at different locations of collagen cause different disruptions of the triple helix. Here, we describe markedly different conformational changes of the triple helix induced by two Gly substitution mutations placed only 12 residues apart. The effects of the Gly substitutions were characterized using a recombinant collagen fragment modeling the 63-residue segment of the ␣1 chain of type I collagen containing no Hyp (residues 877-939) obtained from Escherichia coli. Two Gly 3 Ser substitutions at Gly-901 and Gly-913 associated with, respectively, mild and severe OI variants were introduced by site-directed mutagenesis. Biophysical characterization and limited protease digestion experiments revealed that while the substitution at Gly-901 causes relatively minor destabilization of the triple helix, the substitution at Gly-913 induces large scale unfolding of an unstable region C-terminal to the mutation site. This extensive unfolding is caused by the intrinsic low stability of the C-terminal region of the helix and the mutation induced disruption of a set of salt bridges, which functions to lock this unstable region into the triple helical conformation. The extensive conformational changes associated with the loss of the salt bridges highlight the long range impact of the local interactions of triple helix and suggest a new mechanism by which OI mutations cause severe conformational damages in collagen.Considerable effort has been made to elucidate the mechanisms by which Gly substitution mutations of the collagen triple helix cause Osteogenesis Imperfecta (OI), 2 also known as brittle bone disease. The collagen triple helix consists of three polypeptide chains each in extended polyproline II conformation and with the characteristic (Gly-X-Y) n repeating amino acid sequence (1-3). The Gly at every third position is necessitated by the close packing of the helix; while the X and Y residues (where X and Y can be any amino acids) contribute directlytothestabilityofthetriplehelixandconferthesequencedependent properties of collagen (4). Missense mutations that replace the obligatory Gly by another amino acid residue in type I collagen, the major component of bones, are the most common cause of OI (5, 6). The triple helix domain of type I collagen is a heterotrimer composed of two ␣1 chains and one ␣2 chain each with more than 1000 amino acids in an uninterrupted (Gly-X-Y) n sequence (7). Nearly 800 Gly replacing mutations from both ␣1 and ␣2 chains have been linked to OI, yet, depending on the location and the identity of the Gly substitution, the clinical severity of OI varies from mild increase of bone fragility to the most severe type characterized by death at the prenatal stage (the Type II OI) (6). It remains unclear what molecular properties are related to the se...

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