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Reflections on the catalytic power of a TIM-barrel.

ABSTRACT: The TIM-barrel fold is described and its propagation throughout the enzyme universe noted. The functions of the individual front loops of the eponymous TIM-barrel of triosephosphate isomerase are presented in a discussion of: (a) electrophilic catalysis, by amino acid side chains from loops 1 and 4, of abstraction of an ?-carbonyl hydrogen from substrate dihydroxyacetone phosphate (DHAP) or d-glyceraldehyde 3-phosphate (DGAP). (b) The engineering of loop 3 to give the monomeric variant monoTIM and the structure and catalytic properties of this monomer. (c) The interaction between loops 6, 7 and 8 and the phosphodianion of DHAP or DGAP. (d) The mechanism by which a ligand-gated conformational change, dominated by motion of loops 6 and 7, activates TIM for catalysis of deprotonation of DHAP or DGAP. (e) The conformational plasticity of TIM, and the utilization of substrate binding energy to "mold" the distorted active site loops of TIM mutants into catalytically active enzymes. The features of the TIM-barrel fold that favor effective protein catalysis are discussed.

SUBMITTER: Richard JP

PROVIDER: S-EPMC4256097 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Reflections on the catalytic power of a TIM-barrel.

Richard John P JP Zhai Xiang X Malabanan M Merced MM

Bioorganic chemistry 20140711

The TIM-barrel fold is described and its propagation throughout the enzyme universe noted. The functions of the individual front loops of the eponymous TIM-barrel of triosephosphate isomerase are presented in a discussion of: (a) electrophilic catalysis, by amino acid side chains from loops 1 and 4, of abstraction of an α-carbonyl hydrogen from substrate dihydroxyacetone phosphate (DHAP) or d-glyceraldehyde 3-phosphate (DGAP). (b) The engineering of loop 3 to give the monomeric variant monoTIM a ...[more]

PMID: 25092608

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Project description: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.

Project description:Exploring the potential functions of nonconserved residues on the outer side of α-helices and systematically optimizing them are pivotal for their application in protein engineering. Based on the evolutionary structural conservation analysis of GH5_5 cellulases, a practical molecular improvement strategy was developed. Highly variable sites on the outer side of the α-helices of the GH5_5 cellulase from Aspergillus niger (AnCel5A) were screened, and 14 out of the 34 highly variable sites were confirmed to exert a positive effect on the activity. After the modular combination of the positive mutations, the catalytic efficiency of the mutants was further improved. By using CMC-Na as the substrate, the catalytic efficiency and specific activity of variant AnCel5A_N193A/T300P/D307P were approximately 2.0-fold that of AnCel5A (227 ± 21 versus 451 ± 43 ml/s/mg and 1,726 ± 19 versus 3,472 ± 42 U/mg, respectively). The half-life (t1/2) of variant AnCel5A_N193A/T300P/D307P at 75°C was 2.36 times that of AnCel5A. The role of these sites was successfully validated in other GH5_5 cellulases. Computational analyses revealed that the flexibility of the loop 6-loop 7-loop 8 region was responsible for the increased catalytic performance. This work not only illustrated the important role of rapidly evolving positions on the outer side of the α-helices of GH5_5 cellulases but also revealed new insights into engineering the proteins that nature left as clues for us to find. IMPORTANCE A comprehensive understanding of the residues on the α-helices of the GH5_5 cellulases is important for catalytic efficiency and stability improvement. The main objective of this study was to use the evolutionary conservation and plasticity of the TIM-barrel fold to probe the relationship between nonconserved residues on the outer side of the α-helices and the catalytic efficiency of GH5_5 cellulases by conducting structure-guided protein engineering. By using a four-step nonconserved residue screening strategy, the functional role of nonconserved residues on the outer side of the α-helices was effectively identified, and a variant with superior performance and capability was constructed. Hence, this study proved the effectiveness of this strategy in engineering GH5_5 cellulases and provided a potential competitor for industrial applications. Furthermore, this study sheds new light on engineering TIM-barrel proteins.

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Reflections on the catalytic power of a TIM-barrel.

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Reflections on the catalytic power of a TIM-barrel.

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