ArticleBMC biochemistry2015
Design of symmetric TIM barrel proteins from first principles.
Article in BMC biochemistry, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed.
- Designing de novo TIM barrels: insights into stabilization, diversification, and functionalization strategies.Biochemical Society transactions · 2026Review
- Article
- The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.Journal of molecular biology · 2021Article
- Evolution, folding, and design of TIM barrels and related proteins.Current opinion in structural biology · 2021Review
- Expanded analyses of the functional correlations within structural classifications of glycoside hydrolases.Computational and structural biotechnology journal · 2021Article
- Hydrogen-Deuterium Exchange within Adenosine Deaminase, a TIM Barrel Hydrolase, Identifies Networks for Thermal Activation of Catalysis.Journal of the American Chemical Society · 2020Article
- An allosteric pathway explains beneficial fitness in yeast for long-range mutations in an essential TIM barrel enzyme.Protein science : a publication of the Protein Society · 2020Article
- Development and applications of artificial symmetrical proteins.Computational and structural biotechnology journal · 2020Review
- Ω76: A designed antimicrobial peptide to combat carbapenem- and tigecycline-resistantScience advances · 2019Article
- SAMbinder: A Web Server for Predicting S-Adenosyl-L-Methionine Binding Residues of a Protein From Its Amino Acid Sequence.Frontiers in pharmacology · 2019Article
- Design of a heme-binding peptide motif adopting a β-hairpin conformation.The Journal of biological chemistry · 2018Article
- Correlation of fitness landscapes from three orthologous TIM barrels originates from sequence and structure constraints.Nature communications · 2017Article
- Unveiling the functional diversity of the alpha/beta hydrolase superfamily in the plant kingdom.Current opinion in structural biology · 2016Review
- Protein Design: Getting to the bottom of the TIM barrel.Nature chemical biology · 2016Article
- De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy.Nature chemical biology · 2016Article
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Authors and funding
3 authors.
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No grant is acknowledged in the PubMed record.
Abstract
backgroundComputational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (α/β)8 TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts.
methodsComputational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics.
resultsExtensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a Tm of 44 °C and a Gibbs free energy of unfolding (ΔG°) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a Cm of 1.6 M and a ΔG° of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics.
conclusionsDespite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.
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