A Diketoreductaseexhibits Unique Renaturation Profile from Thermal-Induced Protein Unfolding

Amino Acids

A DiketoreductaseExhibits Unique Renaturation Profile from Thermal-induced Protein Unfolding

Meiling Lu1.Xu Cao2.Xin Yang1. Heng Zheng2.Nan Liu1.

Yun Jiang1. Donghai Lin2,3.Yijun Chen1

1Laboratory of Chemical Biology, China Pharmaceutical University ,24 Tongjia Street, Nanjing, Jiangsu Province, 210009, People’s Republic of China

2School of Life Science and Technology, China Pharmaceutical University, 24 Tongjia Street, Nanjing, Jiangsu Province, 210009, People’s Republic of China

3Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, People’s Republic of China

Email:

Supplementary Table1. Effects of protein concentration on the activity recoverya.

Concentration (mg ml-1) / 25 ºC
(U mg-1)b / 60 ºC
(U mg-1) / 70 ºC
(U mg-1) / 80 ºC
(U mg-1) / 90 ºC
(U mg-1)
0.05 / 8.87 / 8.84 / 7.47 / 7.60 / 8.02
0.10 / 8.89 / 6.43 / 6.20 / 6.43 / 7.06
0.15 / 8.78 / 4.11 / 3.20 / 2.99 / 3.40
0.20 / 8.83 / 2.08 / 1.07 / 0.76 / 1.91

aDifferent concentrations of rDKR were treated at various temperatures, and then cooled at 25 ºC for 1 h. Enzyme activity was measured after cooling.

bSpecific activity of rDKR.

Supplementary Table2. Secondary structural elements of rDKR at different temperatures by CD analysis.

25ºC / 60ºC / 70ºC / 80ºC / 90ºC
Native / Unfolded / Renatured / Unfolded / Renatured / Unfolded / Renatured / Unfolded / Renatured
α-helix / 45.7% / 27.3% / 27.4% / 22.9% / 31.8% / 20.3% / 39.2% / 19.0% / 42.9%
β-sheet / 18.3% / 19.9% / 33.8% / 28.4% / 28.4% / 30.0% / 23.9% / 26.1% / 19.8%
Turn / 15.0% / 15.4% / 10.3% / 12.3% / 11.0% / 11.5% / 13.5% / 13.7% / 15.2%
Random / 21.0% / 23.6% / 28.6% / 36.4% / 28.8% / 38.2% / 23.5% / 41.2% / 22.1%

1

Supplementary Fig. 1Correlation between enzymatic activity and molar ellipticity

solid circles: activity assay from 55 to 65ºC with 1ºC interval; open circles: molar ellipticity at 222nmfrom 55 to 65ºC with 1ºC interval.

Supplementary Fig. 2Far-UV CD spectra of rDKR from 25 to 90ºC

CD spectra are the overlay of rDKR from 25 to 90 ºC with temperature increased stepwise by 5ºC increments. The data are averages of three scans.

Supplementary Fig. 3Far-UV CD spectra of rDKR on the renaturation at different denaturing temperatures

(A) CD spectra of rDKR unfolded and renatured from 90 ºC. (B) CD spectra of rDKR unfolded and renatured from 60 ºC. (C) CD spectra of rDKR unfolded and renatured from 70 ºC.(D)CD spectra of rDKR unfolded and renatured from 80 ºC. black line: native; red line: unfolded; green line: renatured.

Supplementary Fig. 4 Far-UV CD spectra of rDKR from 90 to 25 ºC

CD spectra are the overlay of rDKR from 90 to 25 ºC with temperature decreased stepwise by 5ºC increments. The cooling rate was 1 ºC min-1.

Supplementary Fig. 5UV spectra of rDKR at different temperatures

UV spectra were scanned from 25 to 60 ºC with stepwise by 5ºC increments. Protein concentration was 0.2 mgml-1.

1