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Thermostable artificial enzyme isolated by in vitro selection.

Morelli A, Haugner J, Seelig B - PLoS ONE (2014)

Bottom Line: This process commonly results in a simultaneous reduction of protein stability as an undesired side effect.Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes.These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America, & BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, United States of America.

ABSTRACT
Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65 °C. The most efficient enzyme, ligase 10 C, was not only active at 65 °C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10 C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

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Sequence differences between ligase #7 and ligase 10C mapped onto the NMR structure of ligase 10C [20].Mutations are shown in red. Residues potentially perturbed by the mutations are labeled in blue and long range NOEs are shown as dashed black lines. The two coordinated zinc ions as depicted as orange spheres and the residue numbers refer to ligase 10C. The unstructured termini of ligase 10C were omitted for clarity.
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pone-0112028-g006: Sequence differences between ligase #7 and ligase 10C mapped onto the NMR structure of ligase 10C [20].Mutations are shown in red. Residues potentially perturbed by the mutations are labeled in blue and long range NOEs are shown as dashed black lines. The two coordinated zinc ions as depicted as orange spheres and the residue numbers refer to ligase 10C. The unstructured termini of ligase 10C were omitted for clarity.

Mentions: The increased thermostability of ligase 10C was likely due to additional intramolecular contacts within the protein compared to the mesophilic ligases #6 and #7. In contrast to these enzymes isolated at 23°C, the properties of ligase variant 10C were suitable to solve its three-dimensional solution structure by NMR [20]. The structure featured a small, well-folded core coordinated by two Zn2+-ions. In addition, the folding core also contained a highly dynamic internal loop and was framed by unstructured termini. In order to discuss a potential correlation between differences in primary sequence and altered thermal stability, we mapped sequence differences between ligase #7 and 10C onto the structure of 10C (Figure 6). We chose ligase #7 for comparison because despite the high sequence similarity it showed a large difference in thermostability. All sequence differences between these two ligases were found in or near the structured region responsible for zinc coordination. We previously demonstrated by NMR that residues Ile68 and His69 near the C-terminus of ligase 10C made long range NOE contacts with several residues at the N-terminus (Lys17, His18, Ala27 and Glu28) [20]. Notably, His18 was one of the zinc coordinating residues in ligase 10C [20] and mutating this position to Ala resulted in a drastically reduced solubility of the protein. In ligase #7, the residue corresponding to Ile68 was a methionine. In addition, ligase #7 contained an additional 13 amino acids located between the residues corresponding to Ile68 and His69 in ligase 10C, which likely moved His69 and prevented its contacts with Lys17, His18 at the N-terminus. Presumably, all these mutations could compromise the intramolecular interactions in these positions reported for ligase 10C and decrease the stability of ligase #7 at high temperature. Ligase 10C also differed from ligase #7 in two additional positions (Ser54 and Asp65) which may further influence protein stability. A direct comparison of the overall flexibility of ligase 10C and the two mesophilic ligases would require solving also the structures of ligases #6 and #7 by NMR. This would be beyond the scope of this paper and preliminary experiments suggested that ligase #6 is not amenable to detailed NMR studies.


Thermostable artificial enzyme isolated by in vitro selection.

Morelli A, Haugner J, Seelig B - PLoS ONE (2014)

Sequence differences between ligase #7 and ligase 10C mapped onto the NMR structure of ligase 10C [20].Mutations are shown in red. Residues potentially perturbed by the mutations are labeled in blue and long range NOEs are shown as dashed black lines. The two coordinated zinc ions as depicted as orange spheres and the residue numbers refer to ligase 10C. The unstructured termini of ligase 10C were omitted for clarity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230948&req=5

pone-0112028-g006: Sequence differences between ligase #7 and ligase 10C mapped onto the NMR structure of ligase 10C [20].Mutations are shown in red. Residues potentially perturbed by the mutations are labeled in blue and long range NOEs are shown as dashed black lines. The two coordinated zinc ions as depicted as orange spheres and the residue numbers refer to ligase 10C. The unstructured termini of ligase 10C were omitted for clarity.
Mentions: The increased thermostability of ligase 10C was likely due to additional intramolecular contacts within the protein compared to the mesophilic ligases #6 and #7. In contrast to these enzymes isolated at 23°C, the properties of ligase variant 10C were suitable to solve its three-dimensional solution structure by NMR [20]. The structure featured a small, well-folded core coordinated by two Zn2+-ions. In addition, the folding core also contained a highly dynamic internal loop and was framed by unstructured termini. In order to discuss a potential correlation between differences in primary sequence and altered thermal stability, we mapped sequence differences between ligase #7 and 10C onto the structure of 10C (Figure 6). We chose ligase #7 for comparison because despite the high sequence similarity it showed a large difference in thermostability. All sequence differences between these two ligases were found in or near the structured region responsible for zinc coordination. We previously demonstrated by NMR that residues Ile68 and His69 near the C-terminus of ligase 10C made long range NOE contacts with several residues at the N-terminus (Lys17, His18, Ala27 and Glu28) [20]. Notably, His18 was one of the zinc coordinating residues in ligase 10C [20] and mutating this position to Ala resulted in a drastically reduced solubility of the protein. In ligase #7, the residue corresponding to Ile68 was a methionine. In addition, ligase #7 contained an additional 13 amino acids located between the residues corresponding to Ile68 and His69 in ligase 10C, which likely moved His69 and prevented its contacts with Lys17, His18 at the N-terminus. Presumably, all these mutations could compromise the intramolecular interactions in these positions reported for ligase 10C and decrease the stability of ligase #7 at high temperature. Ligase 10C also differed from ligase #7 in two additional positions (Ser54 and Asp65) which may further influence protein stability. A direct comparison of the overall flexibility of ligase 10C and the two mesophilic ligases would require solving also the structures of ligases #6 and #7 by NMR. This would be beyond the scope of this paper and preliminary experiments suggested that ligase #6 is not amenable to detailed NMR studies.

Bottom Line: This process commonly results in a simultaneous reduction of protein stability as an undesired side effect.Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes.These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America, & BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, United States of America.

ABSTRACT
Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65 °C. The most efficient enzyme, ligase 10 C, was not only active at 65 °C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10 C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

Show MeSH