A high throughput single-molecule method for identifying peptides and sequencing proteins based on nanopores could reduce costs and increase speeds of sequencing, allow the fabrication of portable ...home-diagnostic devices, and permit the characterization of low abundance proteins and heterogeneity in post-translational modifications. Here we engineer the size of Fragaceatoxin C (FraC) biological nanopore to allow the analysis of a wide range of peptide lengths. Ionic blockades through engineered nanopores distinguish a variety of peptides, including two peptides differing only by the substitution of alanine with glutamate. We also find that at pH 3.8 the depth of the peptide current blockades scales with the mass of the peptides irrespectively of the chemical composition of the analyte. Hence, this work shows that FraC nanopores allow direct readout of the mass of single peptide in solution, which is a crucial step towards the developing of a real-time and single-molecule protein sequencing device.
The modular structure of many protein families, such as β-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate ...forms. Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical β-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2–10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry.
Significance In this study, we have designed and experimentally validated, to our knowledge, the first perfectly symmetrical β-propeller protein. Our results provide insight not only into protein evolution through duplication events, but also into methods for creating designer proteins that self-assemble according to simple arithmetical rules. Such proteins may have very wide uses in bionanotechnology. Furthermore our design approach is both rapid and applicable to many different protein templates. Our novel propeller protein consists of six identical domains known as “blades.” Using a variety of biophysical techniques, we show it to be highly stable and report several high-resolution crystal structures of different forms of the protein. Domain swapping allows us to generate related oligomeric forms with fixed numbers of blades per complex.
We have engineered a metal‐binding site into the novel artificial β‐propeller protein Pizza. This new Pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with ...three‐fold symmetry. The designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. Two copies of the trimer associate in the presence of cadmium chloride in solution, and very high‐resolution X‐ray crystallographic analysis reveals a nanocrystal of cadmium chloride, sandwiched between two trimers of the protein. This nanocrystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. Our results indicate the feasibility of using rationally designed symmetrical proteins to biomineralize nanocrystals with useful properties.
Protein design: A novel artificial protein with pseudo‐six‐fold symmetry creates a nanocrystal of cadmium chloride, sandwiched between two copies of the protein (see picture). X‐ray structural evidence for the formation of the nanocrystal inside the protein is presented.
Saccharomyces cerevisiae (S. cerevisiae)invertase is encoded by a family of closely related SUC genes. To identify and understand the molecular basis for differences in substrate specificity, we ...examined 29 SUC alleles from industrialS. cerevisiaestrains and cloned alleles with small sequence differences into an invertase-negative strain. Our study showed that an F102Y substitution in Suc-enzymes lowers yeast invertase activity toward fructo-oligosaccharides (FOS) by 36% and the specificity factor by 43%. By contrast, an A409P substitution in Suc-enzymes resulted in an increased capacity of the yeast to hydrolyze FOS and Fibruline by 17 and 41%, respectively, likely because of a change in the loop conformation resulting in a wider active site. Bread dough fermentation experiments revealed that sucrose and fructan hydrolysis during fermentation is influenced by this natural variation in SUC sequences. Our research thus opens the door for the selection or engineering of yeasts and Suc-enzymes with specific activities that may ultimately allow controlling fructan hydrolysis.
Novel bioinorganic hybrid materials based on proteins and inorganic clusters have enormous potential for the development of hybrid catalysts that synergistically combine properties of both materials. ...Here we report the creation of hybrid assemblies between a computationally designed symmetrical protein Pizza6-S and different polyoxometalates with matching symmetry: the tellurotungstic Anderson-Evans (Na
6
TeW
6
O
24
·22H
2
O) (TEW); Keggin (H
4
SiW
12
O
40
·6H
2
O) (STA); and 1 : 2 Ce
III
-substituted Keggin (K
11
Ce
III
PW
11
O
39
2
·20H
2
O) (Ce-K). This results in the formation of complexes with clearly defined stoichiometries in solution. Crystal structures validate the complexes as building blocks for the formation of larger assemblies.
A symmetric designer protein forms hybrid complexes with different polyoxometalates and may serve as a building block for porous frameworks.
Formula: see text-Propeller proteins are common natural disc-like pseudo-symmetric proteins that contain multiple repeats ('blades') each consisting of a 4-stranded anti-parallel Formula: see ...text-sheet. So far, 4- to 12-bladed Formula: see text-propellers have been discovered in nature showing large functional and sequential variation. Using computational design approaches, we created perfectly symmetric Formula: see text-propellers out of natural pseudo-symmetric templates. These proteins are useful tools to study protein evolution of this very diverse fold. While the 7-bladed architecture is the most common, no symmetric 7-bladed monomer has been created and characterized so far. Here we describe such a engineered protein, based on a highly symmetric natural template, and test the effects of circular permutation on its stability. Geometrical analysis of this protein and other artificial symmetrical proteins reveals no systematic constraint that could be used to help in engineering of this fold, and suggests sequence constraints unique to each Formula: see text-propeller sub-family.
Hierarchical self-assembly of hybrid bioinorganic structures is a challenging task which requires specific and tailored interactions. Here we report a supramolecular assembly formed between the ...six-bladed symmetrical designer protein Pizza6-S (Pizza6) and the {K3Cu3(NO3)A-α-PW9O342} (Cu3) polyoxometalate (POM). The crystal structure (1.8 Å resolution) revealed that the Cu3 dissociated and reassembled with the protein to form a novel POM-protein cage. In this hybrid assembly, six CuII ions link two Pizza6 molecules in a controlled way by binding to the six symmetrically equivalent histidine side chains. Such coordination results in the formation of a “bioinorganic cage” in which a lacunary A-α-PW9O349– (PW9) anion is tightly encapsulated via coordination to CuII ions and hydrogen bonding with protein side chains. Further spectroscopic characterization of the Pizza6/Cu3 solution suggests that dissociation of Cu3 is facilitated by the synergetic effect of six histidine residues which have high affinity toward Cu(II) ions, resulting in the formation of the hierarchical supramolecular assembly.
Artificial β-propeller protein-based hydrolases Clarke, David E; Noguchi, Hiroki; Gryspeerdt, Jean-Louis A. G ...
Chemical communications (Cambridge, England),
07/2019, Letnik:
55, Številka:
6
Journal Article
Recenzirano
Odprti dostop
We developed an artificial hydrolase based on the symmetrical Pizza6 β-propeller protein for the metal-free hydrolysis of 4-nitrophenyl acetate and butyrate. Through site-specific mutagenesis and ...crystallisation studies, the catalytic mechanism was investigated and found to be dependent on a threonine-histidine dyad. The mutant with additional histidine residues generated the highest
k
cat
values, forming a His-His-Thr triad and matched previously reported metalloenzymes. The highly symmetrical β-propeller artificial enzymes and their protein-metal complexes have potential to be utilised in bioinorganic and supramolecular chemistry, as well as being developed further into 2D/3D catalytic materials.
We investigated symmetrical β-propeller protein scaffolds as artificial hydrolases and discovered their catalytic mechanism to be centred around a threonine-histidine dyad.
β‐propeller proteins are common in nature, where they are observed to adopt 4‐ to 10‐fold internal rotational pseudo‐symmetry. This size diversity can be explained by the evolutionary process of gene ...duplication and fusion. In this study, we investigated a distorted β‐propeller protein, an apparent intermediate between two symmetries. From this template, we created a perfectly symmetric 9‐bladed β‐propeller named Cake, using computational design and ancestral sequence reconstruction. The designed repeat sequence was found to be capable of generating both 8‐fold and 9‐fold propellers which are highly stable. Cake variants with 2–10 identical copies of the repeat sequence were characterised by X‐ray crystallography and in solution. They were found to be highly stable, and to self‐assemble into 8‐ or 9‐fold symmetrical propellers. These findings show that the β‐propeller fold allows sufficient structural plasticity to permit a given blade to assemble different forms, a transition from even to odd changes in blade number, and provide a potential explanation for the wide diversity of repeat numbers observed in natural propeller proteins.
Database
Structural data are available in Protein Data Bank database under the accession numbers 6TJB, 6TJC, 6TJD, 6TJE, 6TJF, 6TJG, 6TJH and 6TJI.
Computational protein design was applied on a distorted β‐propeller resulting in a perfectly symmetrical 9‐bladed propeller. The designed sequence was also able to fold as an 8‐bladed propeller. Our finding illustrates that the β‐propeller fold allows structural plasticity explaining the variety of repeats found in nature for these proteins.
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