Selection and structural analysis of de novo proteins from an alpha3beta3 genetic library.
ABSTRACT: The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding alpha3beta3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP-based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8-anilino-1-naphthalenesulfonate (ANS) binding assay, and analytical size-exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both alpha-helix and beta-sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten-globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three alpha-helices and three beta-strands as intended by its design. Thus, it would appear that artificially generated alpha3beta3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.
Project description:Many single-domain proteins are not only stable and water-soluble, but they also populate few to no intermediates during folding. This reduces interactions between partially folded proteins, misfolding, and aggregation, and makes the proteins tractable in biotechnological applications. Natural proteins fold thus, not necessarily only because their structures are well-suited for folding, but because their sequences optimize packing and fit their structures well. In contrast, folding experiments on the de novo designed Top7 suggest that it populates several intermediates. Additionally, in de novo protein design, where sequences are designed for natural and new non-natural structures, tens of sequences still need to be tested before success is achieved. Both these issues may be caused by the specific scaffolds used in design, i.e., some protein scaffolds may be more tolerant to packing perturbations and varied sequences. Here, we report a computational method for assessing the response of protein structures to packing perturbations. We then benchmark this method using designed proteins and find that it can identify scaffolds whose folding gets disrupted upon perturbing packing, leading to the population of intermediates. The method can also isolate regions of both natural and designed scaffolds that are sensitive to such perturbations and identify contacts which when present can rescue folding. Overall, this method can be used to identify protein scaffolds that are more amenable to whole protein design as well as to identify protein regions which are sensitive to perturbations and where further mutations should be avoided during protein engineering.
Project description:Natural recombination combines pieces of preexisting proteins to create new tertiary structures and functions. We describe a computational protocol, called SEWING, which is inspired by this process and builds new proteins from connected or disconnected pieces of existing structures. Helical proteins designed with SEWING contain structural features absent from other de novo designed proteins and, in some cases, remain folded at more than 100°C. High-resolution structures of the designed proteins CA01 and DA05R1 were solved by x-ray crystallography (2.2 angstrom resolution) and nuclear magnetic resonance, respectively, and there was excellent agreement with the design models. This method provides a new strategy to rapidly create large numbers of diverse and designable protein scaffolds.
Project description:Repeat proteins have considerable potential for use as modular binding reagents or biomaterials in biomedical and nanotechnology applications. Here we describe a general computational method for building idealized repeats that integrates available family sequences and structural information with Rosetta de novo protein design calculations. Idealized designs from six different repeat families were generated and experimentally characterized; 80% of the proteins were expressed and soluble and more than 40% were folded and monomeric with high thermal stability. Crystal structures determined for members of three families are within 1Å root-mean-square deviation to the design models. The method provides a general approach for fast and reliable generation of stable modular repeat protein scaffolds.
Project description:Isopeptide bond-tethered triple-stranded coiled coils of HIV-1 gp41 N-terminal heptad repeat (NHR) peptides have been designed with de novo auxiliaries to guide site-directed trimerized cross-linking. The presence of isopeptide bridges in the rationally designed trimerization motifs provides extraordinary stability to withstand thermal and chemical denaturation. As a result, these ultra-stable and well-folded trimeric coiled coils direct and yield proteolysis-resistant and remarkably potent N-peptide chimeric trimers with HIV-1 fusion inhibitory activities in the low nanomolar range, much more effective than the corresponding unstructured N-peptide monomers and reaching the potency of clinically used T20 peptide (enfuvirtide). Thus, these isopeptide bond-crosslinked de novo coiled coils may also be used as attractive scaffolds for isolating NHR-trimers in other class I enveloped viruses for therapeutic intervention. Furthermore, this isopeptide bridge-tethering strategy could be extendable to the construction of ultra-stable proteins interfering with certain biological processes.
Project description:Protein nanostructures with precisely defined geometries have many potential applications in catalysis, sensing, signal processing, and drug delivery. While many de novo protein nanostructures have been assembled via non-covalent intramolecular and intermolecular interactions, a largely unexplored strategy is to construct nanostructures by covalently linking multiple individually folded proteins through site-specific ligations. Here, we report the synthesis of single-chain protein nanostructures with triangular and square shapes made using multiple copies of a three-helix bundle protein and split intein chemistry. Coarse-grained simulations confirm the experimentally observed flexibility of these nanostructures, which is optimized to produce triangular structures with high regularity. These single-chain nanostructures also display ultra-high thermostability, resist denaturation by chaotropes and organic solvents, and have applicability as scaffolds for assembling materials with nanometer resolution. Our results show that site-specific covalent ligation can be used to assemble individually folded proteins into single-chain nanostructures with bespoke architectures and high stabilities.
Project description:Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.
Project description:Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12?°C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25?°C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.
Project description:Liquid-liquid phase separation (LLPS) of proteins that leads to formation of membrane-less organelles is critical to many biochemical processes in the cell. However, dysregulated LLPS can also facilitate aberrant phase transitions and lead to protein aggregation and disease. Accordingly, there is great interest in identifying small molecules that modulate LLPS. Here, we demonstrate that 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and similar compounds are potent biphasic modulators of protein LLPS. Depending on context, bis-ANS can both induce LLPS de novo as well as prevent formation of homotypic liquid droplets. Our study also reveals the mechanisms by which bis-ANS and related compounds modulate LLPS and identify key chemical features of small molecules required for this activity. These findings may provide a foundation for the rational design of small molecule modulators of LLPS with therapeutic value.
Project description:Zoysiagrass (Zoysia japonica Steud.) is commonly found in temperate climate regions and widely used for lawns, in part, owing to its uniform green color. However, some zoysiagrass cultivars accumulate red to purple pigments in their spike and stolon tissues, thereby decreasing the aesthetic value. Here we analyzed the anthocyanin contents of two zoysiagrass cultivars 'Anyang-jungji' (AJ) and 'Greenzoa' (GZ) that produce spikes and stolons with purple and green colors, respectively, and revealed that cyanidin and petunidin were primarily accumulated in the pigmented tissues. In parallel, we performed a de novo transcriptome assembly and identified differentially expressed genes between the two cultivars. We found that two anthocyanin biosynthesis genes encoding anthocyanidin synthase (ANS) and dihydroflavonol 4-reductase (DFR) were preferentially upregulated in the purple AJ spike upon pigmentation. Both ANS and DFR genes were also highly expressed in other zoysiagrass cultivars with purple spikes and stolons, but their expression levels were significantly low in the cultivars with green tissues. We observed that recombinant ZjDFR1 and ZjANS1 proteins successfully catalyze the conversions of dihydroflavonols into leucoanthocyanidins and leucoanthocyanidins into anthocyanidins, respectively. These findings strongly suggest that upregulation of ANS and DFR is responsible for tissue-specific anthocyanin biosynthesis and differential pigmentation in zoysiagrass. The present study also demonstrates the feasibility of a de novo transcriptome analysis to identify the key genes associated with specific traits, even in the absence of reference genome information.
Project description:The de novo evolution of protein-coding genes from noncoding DNA is emerging as a source of molecular innovation in biology. Studies of random sequence libraries, however, suggest that young de novo proteins will not fold into compact, specific structures typical of native globular proteins. Here we show that Bsc4, a functional, natural de novo protein encoded by a gene that evolved recently from noncoding DNA in the yeast S. cerevisiae, folds to a partially specific three-dimensional structure. Bsc4 forms soluble, compact oligomers with high ? sheet content and a hydrophobic core, and undergoes cooperative, reversible denaturation. Bsc4 lacks a specific quaternary state, however, existing instead as a continuous distribution of oligomer sizes, and binds dyes indicative of amyloid oligomers or molten globules. The combination of native-like and non-native-like properties suggests a rudimentary fold that could potentially act as a functional intermediate in the emergence of new folded proteins de novo.