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Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: factors contributing to increased activity retention.

ABSTRACT: Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65 °C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.

SUBMITTER: Augustyniak W

PROVIDER: S-EPMC3375749 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: factors contributing to increased activity retention.

Augustyniak Wojciech W Brzezinska Agnieszka A AA Pijning Tjaard T Wienk Hans H Boelens Rolf R Dijkstra Bauke W BW Reetz Manfred T MT

Protein science : a publication of the Protein Society 20120229 4

Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65 °C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical charac ...[more]

PMID: 22267088

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Project description:Proteins in thermophilic organisms remain stable and function optimally at high temperatures. Owing to their important applicability in many industrial processes, such thermostable proteins have been studied extensively, and several structural factors attributed to their enhanced stability. How these factors render the emergent property of thermostability to proteins, even in situations where no significant changes occur in their three-dimensional structures in comparison to their mesophilic counter-parts, has remained an intriguing question. In this study we treat Lipase A from Bacillus subtilis and its six thermostable mutants in a unified manner and address the problem with a combined complex network-based analysis and molecular dynamic studies to find commonality in their properties. The Protein Contact Networks (PCN) of the wild-type and six mutant Lipase A structures developed at a mesoscopic scale were analyzed at global network and local node (residue) level using network parameters and community structure analysis. The comparative PCN analysis of all proteins pointed towards important role of specific residues in the enhanced thermostability. Network analysis results were corroborated with finer-scale molecular dynamics simulations at both room and high temperatures. Our results show that this combined approach at two scales can uncover small but important changes in the local conformations that add up to stabilize the protein structure in thermostable mutants, even when overall conformation differences among them are negligible. Our analysis not only supports the experimentally determined stabilizing factors, but also unveils the important role of contacts, distributed throughout the protein, that lead to thermostability. We propose that this combined mesoscopic-network and fine-grained molecular dynamics approach is a convenient and useful scheme not only to study allosteric changes leading to protein stability in the face of negligible over-all conformational changes due to mutations, but also in other molecular networks where change in function does not accompany significant change in the network structure.

Dataset Information

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: factors contributing to increased activity retention.

Publications

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: factors contributing to increased activity retention.

Similar Datasets

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