What is it about?
To enhance the heat stability of the CutA1 protein from Escherichia coli (EcCutA1) so that it has compar- able stability to CutA1 from Pyrococcus horikoshii with a denaturation temperature (Td) of 150C, we used the Stability Profile of Mutant Protein (SPMP) to examine the structure-sequence (3D-1D) compatibil- ity between the conformation of EcCutA1 and its native sequence [J. Mol. Biol., 248, 733-738, (1995)]. We identified seven residues in EcCutA1 that were incom- patible in terms of dihedral angles and hydrophobicity. These residues were replaced with appropriate amino acids, and the mutant proteins were evaluated for changes in stability by DSC and denaturant denatur- ation. The mutations that were introduced at five out of the seven positions improved the stability of EcCutA1. The Td values of single (S11A) and triple (S11V/E61V/ Q73V) mutants improved by 16.5 and 26.6C, respect- ively, compared to that of the wild-type protein (89.9C). These analyses showed that (1) the stability of EcCutA1 is remarkably improved by slight substitu- tions, even though the stability of the wild-type protein is considerably high, (2) remarkable improvements in the stability can be quantitatively explained based on the newly solved native structure, and (3) SPMP is a powerful tool to examine substitutions that improve protein stability.
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Why is it important?
As a first step in the mutation strategy to enhance the heat stability of the CutA1 protein from EcCutA1, we examined the structure-sequence (3D-1D) compati- bility between the conformation of EcCutA1 and its native sequence using Stability Profile of Mutant Protein (SPMP). SPMP estimates changes in the stability of 19 mutant proteins at every position of a protein based on the X-ray crystal structure (1820). We chose seven incompatible positions in EcCutA1, which were predicted from SPMP, and then introduced single and multiple point mutations at these locations. The stabilities of the constructed EcCutA1 mutants were evaluated by heat and denaturant denaturation, and their structures were determined by X-ray crystal- lography. We will discuss the stabilization mechanism of the mutant proteins based on the X-ray crystalline structure and 3D-1D compatibility.
Perspectives
The stability of the double and triple mutants roughly shows a cumulative effect of each mutant at positions 11, 61 and 73. The Td of the triple mutant, S11V/E61V/ Q73V, was 116.5C, the highest among the examined mutant proteins, and the Td between this triple mutant and the wild-type protein was 26.6C. However, the stabilities of E59K/E61V (104.3C) and S11V/E59K/E61T (112.4 C) were highly similar to E59K (104.2 C) and S11V/E61T (112.3 C), respective- ly. In this case, it seems that the stabilization effect of the mutations at position 61 compensates for removing the electrostatic interaction between Lys 59 and Glu61 due to mutations at Glu61, resulting in no cumulative effect on the overall stability. Overall, the drastic in- creases in stability in the present studies were quantita- tively elucidated based on the native structures, although the denatured structures might be affected by the introduced mutations. Only G45Q and Y86D were remarkably destabilized contrary to the SPMP predictions. Other mutations at these positions were not examined because they are not expected to have greater stability than the wild-type protein.
Dr Bagautdin Bagautdinov
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This page is a summary of: Remarkable improvement in the heat stability of CutA1 from Escherichia coli by rational protein design, The Journal of Biochemistry, July 2010, Oxford University Press (OUP),
DOI: 10.1093/jb/mvq079.
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