Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

Combinatorial assembly of protein domains plays an important role in the evolution of proteins. There is also evidence that protein domains have come together from stable subdomains. This concept of modular assembly could be used to construct new well folded proteins from stable protein fragments. Here, we report the construction of a chimeric protein from parts of a (␤␣)8-barrel enzyme from histidine biosynthesis pathway (HisF) and a protein of the (␤␣)5-flavodoxin-like fold (CheY) from Thermotoga maritima that share a high structural similarity. We expected this construct to fold into a full (␤␣)8-barrel. Our results show that the chimeric protein is a stable monomer that unfolds with high cooperativity. Its three-dimensional structure, which was solved to 3.1 Å resolution by x-ray crystallography, confirms a barrel-like fold in which the overall structures of the parent proteins are highly conserved. The structure further reveals a ninth strand in the barrel, which is formed by residues from the HisF C terminus and an attached tag. This strand invades between ␤-strand 1 and 2 of the CheY part closing a gap in the structure that might be due to a suboptimal fit between the fragments. Thus, by a combination of parts from two different folds and a small arbitrary fragment, we created a well folded and stable protein.chimeric protein ͉ enzyme evolution ͉ flavodoxin-like fold ͉ protein design ͉ TIM-barrel

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confidence: 99%

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confidence: 99%

A βα-barrel built by the combination of fragments from different folds

Bharat

Eisenbeis

Zeth

et al. 2008

“…In earlier work, we investigated the ability of a polypeptide segment to serve as a building block for the creation of folded domains (11). We took a segment from a five-stranded, antiparallel ␤-barrel, the monomeric major cold shock protein (CspA) of Escherichia coli (12,13).…”

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confidence: 99%

A segment of cold shock protein directs the folding of a combinatorial protein

Bono

Riechmann²,

Girard³

et al. 2005

Self Cite

It has been suggested that protein domains evolved by the nonhomologous recombination of building blocks of subdomain size. In earlier work we attempted to recapitulate domain evolution in vitro. We took a polypeptide segment comprising three ␤-strands in the monomeric, five-stranded ␤-barrel cold shock protein (CspA) of Escherichia coli as a building block. This segment corresponds to a complete exon in homologous eukaryotic proteins and includes residues that nucleate folding in CspA. We recombined this segment at random with fragments of natural proteins and succeeded in generating a range of folded chimaeric proteins. We now present the crystal structure of one such combinatorial protein, 1b11, a 103-residue polypeptide that includes segments from CspA and the S1 domain of the 30S ribosomal subunit of E. coli. The structure reveals a segment-swapped, six-stranded ␤-barrel of unique architecture that assembles to a tetramer. Surprisingly, the CspA segment retains its structural identity in 1b11, recapitulating its original fold and deforming the structure of the S1 segment as necessary to complete a barrel. Our work provides structural evidence that (i ) random shuffling of nonhomologous polypeptide segments can lead to folded proteins and unique architectures, (ii ) many structural features of the segments are retained, and (iii ) some segments can act as templates around which the rest of the protein folds. molecular evolution ͉ unique architecture ͉ oligonucleotide͞ oligosaccharide-binding fold

“…Such efforts would benefit the development of in vitro evolution for protein engineering (7)(8)(9) as well. In a recent insightful study, Bogarad and Deem (10) used a generalized (block) NK model, with terms in a potential function (interpreted to represent protein interactions and substrate binding affinities) assigned randomly to a large collection of sequences.…”

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confidence: 99%

Recombinatoric exploration of novel folded structures: A heteropolymer-based model of protein evolutionary landscapes

Cui

Wong

Bornberg‐Bauer

et al. 2002

The role of recombination in evolution is compared with that of point mutations (substitutions) in the context of a simple, polymer physics-based model mapping between sequence (genotype) and conformational (phenotype) spaces. Crossovers and point mutations of lattice chains with a hydrophobic polar code are investigated. Sequences encoding for a single ground-state conformation are considered viable and used as model proteins. Point mutations lead to diffusive walks on the evolutionary landscape, whereas crossovers can ''tunnel'' through barriers of diminished fitness. The degree to which crossovers allow for more efficient sequence and structural exploration depends on the relative rates of point mutations versus that of crossovers and the dispersion in fitness that characterizes the ruggedness of the evolutionary landscape. The probability that a crossover between a pair of viable sequences results in viable sequences is an order of magnitude higher than random, implying that a sequence's overall propensity to encode uniquely is embodied partially in local signals. Consistent with this observation, certain hydrophobicity patterns are significantly more favored than others among fragments (i.e., subsequences) of sequences that encode uniquely, and examples reminiscent of autonomous folding units in real proteins are found. The number of structures explored by both crossovers and point mutations is always substantially larger than that via point mutations alone, but the corresponding numbers of sequences explored can be comparable when the evolutionary landscape is rugged. Efficient structural exploration requires intermediate nonextreme ratios between point-mutation and crossover rates.crossovers ͉ neutral nets ͉ sequence space ͉ thermodynamic stability ͉ lattice protein models I t is widely recognized that key events in evolution may involve large-scale genomic rearrangements (1-6). Experiments on plants suggest that dramatic restructuring of the genome in response to traumas may underlie formations of many new species (1). The presence of introns in the genes of higher organisms implies that even a single base change can result in the deletion or insertion of whole sequences in the protein product (2). It has been argued that cellular ''natural genetic engineering'' machineries have evolved to modulate genomic reorganization in lower organisms (3). Moreover, certain peculiarities in present-day genomes and cellular organizations may be explained best by ''lateral'' or ''horizontal'' transfers in the past (4, 5). Indeed, it is large-scale genomic rearrangements rather than the accumulation of point mutations that bear the main responsibility for the alarmingly quick emergence of bacterial antibiotic resistance.Therefore, to capture evolutionary complexities better, theoretical perspectives that focus exclusively on point mutations should be augmented to include other types of sequence transformations. Such efforts would benefit the development of in vitro evolution for protein engineering (7-9) as well. In a ...