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. Author manuscript; available in PMC: 2020 Mar 26.
Published in final edited form as: Biochemistry. 2019 Mar 7;58(12):1660–1671. doi: 10.1021/acs.biochem.9b00068

Figure 2. In vitro folding of the KvAP channel is dependent on temperature.

Figure 2.

(A, B) Time course of the in vitro folding of the KvAP channel at 20 °C (A) and at 80 °C (B). Representative SDS-PAGE gels showing glutaraldehyde crosslinking of the in vitro folding reaction at various time points over 2 hours is shown. The last lane is a time point at 2 hours without glutaraldehyde added (−). The oligomeric nature of the crosslinked band is indicated. (C) Tetramerization kinetics of the KvAP channel at different temperatures are plotted. Data have been fitted with either a single (40 °C and 80 °C) or a double exponential (50 °C). Data at 20 °C could not be fitted to an exponential. (D) Temperature downshift arrests folding of KvAP. The in vitro folding reaction for the KvAP channel was carried out at 50 °C. After incubation for 2, 5 or 16 minutes, the samples were transferred from 50 °C to 20 °C and the tetramerization kinetics were followed. No additional tetramerization was observed at 20 °C. The data points following the downshift are fitted with a straight line. Error bars shown in C and D are standard deviations for n =3 using at least 2 independent protein preparations.