Limits...
Impact of the 237th residue on the folding of human carbonic anhydrase II.

Wu MJ, Jiang Y, Yan YB - Int J Mol Sci (2011)

Bottom Line: Among the many mutations, the P237H mutation has been characterized to lead to a significant decrease in the activity of the enzyme and in the Gibbs free energy of folding.The FoldX theoretical calculations suggested that this residue did not significantly contribute to the overall folding of HCAII, since all mutants had small ΔΔG values (around 1 kcal/mol).The discrepancy between theoretical and experimental results suggested that caution should be taken when using the prediction methods to evaluate the details of disease-related mutations.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Bio-Resources and Eco-Environment of MOE, College of Life Science, Sichuan University, Chengdu 610064, China; E-Mail: wmj213@gmail.com.

ABSTRACT
The deficiency of human carbonic anhydrase II (HCAII) has been recognized to be associated with a disease called CAII deficiency syndrome (CADS). Among the many mutations, the P237H mutation has been characterized to lead to a significant decrease in the activity of the enzyme and in the Gibbs free energy of folding. However, sequence alignment indicated that the 237th residue of CAII is not fully conserved across all species. The FoldX theoretical calculations suggested that this residue did not significantly contribute to the overall folding of HCAII, since all mutants had small ΔΔG values (around 1 kcal/mol). The experimental determination indicated that at least three mutations affect HCAII folding significantly and the P237H mutation was the most deleterious one, suggesting that Pro237 was important to HCAII folding. The discrepancy between theoretical and experimental results suggested that caution should be taken when using the prediction methods to evaluate the details of disease-related mutations.

Show MeSH

Related in: MedlinePlus

Unfolding transition curves of HCAIIpwt and the mutants monitored by the maximum wavelength of the intrinsic Trp fluorescence. The raw data were fitted by a three-state model and presented as solid lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3116157&req=5

f4-ijms-12-02797: Unfolding transition curves of HCAIIpwt and the mutants monitored by the maximum wavelength of the intrinsic Trp fluorescence. The raw data were fitted by a three-state model and presented as solid lines.

Mentions: HCAIIpwt, which contains a C206S mutation to avoid the interference of unexpected disulfide formation, was used in this study. Previous studies have shown that HCAIIpwt have indistinguishable folding and functional properties from the wild type protein [27–31]. All recombinant proteins could be successfully obtained in the soluble fraction when overexpressed in E. coli. The activities of the mutants were similar to HCAIIpwt, ranging from 87% to 99% of the activity of HCAIIpwt (Table 1). The effect of the mutations on HCAII structure was investigated by circular dichrosim (CD) (Figure 3) and intrinsic fluorescence (data not shown, see also Figure 4) experiments. The spectra of the mutants were almost superimposed with those of HCAIIpwt, suggesting that the mutations did not affect either the secondary or the tertiary structures of HCAIIpwt.


Impact of the 237th residue on the folding of human carbonic anhydrase II.

Wu MJ, Jiang Y, Yan YB - Int J Mol Sci (2011)

Unfolding transition curves of HCAIIpwt and the mutants monitored by the maximum wavelength of the intrinsic Trp fluorescence. The raw data were fitted by a three-state model and presented as solid lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3116157&req=5

f4-ijms-12-02797: Unfolding transition curves of HCAIIpwt and the mutants monitored by the maximum wavelength of the intrinsic Trp fluorescence. The raw data were fitted by a three-state model and presented as solid lines.
Mentions: HCAIIpwt, which contains a C206S mutation to avoid the interference of unexpected disulfide formation, was used in this study. Previous studies have shown that HCAIIpwt have indistinguishable folding and functional properties from the wild type protein [27–31]. All recombinant proteins could be successfully obtained in the soluble fraction when overexpressed in E. coli. The activities of the mutants were similar to HCAIIpwt, ranging from 87% to 99% of the activity of HCAIIpwt (Table 1). The effect of the mutations on HCAII structure was investigated by circular dichrosim (CD) (Figure 3) and intrinsic fluorescence (data not shown, see also Figure 4) experiments. The spectra of the mutants were almost superimposed with those of HCAIIpwt, suggesting that the mutations did not affect either the secondary or the tertiary structures of HCAIIpwt.

Bottom Line: Among the many mutations, the P237H mutation has been characterized to lead to a significant decrease in the activity of the enzyme and in the Gibbs free energy of folding.The FoldX theoretical calculations suggested that this residue did not significantly contribute to the overall folding of HCAII, since all mutants had small ΔΔG values (around 1 kcal/mol).The discrepancy between theoretical and experimental results suggested that caution should be taken when using the prediction methods to evaluate the details of disease-related mutations.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Bio-Resources and Eco-Environment of MOE, College of Life Science, Sichuan University, Chengdu 610064, China; E-Mail: wmj213@gmail.com.

ABSTRACT
The deficiency of human carbonic anhydrase II (HCAII) has been recognized to be associated with a disease called CAII deficiency syndrome (CADS). Among the many mutations, the P237H mutation has been characterized to lead to a significant decrease in the activity of the enzyme and in the Gibbs free energy of folding. However, sequence alignment indicated that the 237th residue of CAII is not fully conserved across all species. The FoldX theoretical calculations suggested that this residue did not significantly contribute to the overall folding of HCAII, since all mutants had small ΔΔG values (around 1 kcal/mol). The experimental determination indicated that at least three mutations affect HCAII folding significantly and the P237H mutation was the most deleterious one, suggesting that Pro237 was important to HCAII folding. The discrepancy between theoretical and experimental results suggested that caution should be taken when using the prediction methods to evaluate the details of disease-related mutations.

Show MeSH
Related in: MedlinePlus