Thermodynamic Study of Phosphoglycerate Kinase from Thermotoga maritima and Its Isolated Domains: Reversible Thermal Unfolding Monitored by Differential Scanning Calorimetry and Circular Dichro
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- 作者：Katrin Zaiss and Rainer Jaenicke
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：April 6, 1999
- 年：1999
- 卷：38
- 期：14
- 页码：4633 - 4639
- 全文大小：114K
- 年卷期：v.38,no.14(April 6, 1999)
- ISSN：1520-4995
文摘
The folding of phosphoglycerate kinase (PGK) from the hyperthermophilic bacteriumThermotoga maritima and its isolated N- and C-terminal domains (N1/2 and C1/2) was characterized bydifferential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. At pH 3.0-4.0,reversible thermal denaturation of TmPGK occurred below 90 
C. The corresponding peaks in the partialmolar heat capacity function were fitted by a four-state model, describing three well-defined unfoldingtransitions. Using CD spectroscopy, these are ascribed to the disruption of the domain interactions andsubsequent sequential unfolding of the two domains. The isolated N-terminal domain unfolds reversiblybetween pH 3.0 and pH 4.0 to >90% and at pH 7.0 to about 70%. In contrast, the isolated engineeredC-terminal domain only shows reversible thermal denaturation between pH 3.0 and pH 3.5. Neither N1/2nor C1/2 obeys the simple two-state mechanism of unfolding. Instead, both unfold via a partially structuredintermediate. In the case of N1/2, the intermediate exhibits native secondary structure and perturbed tertiarystructure, whereas for C1/2 the intermediate could not be defined with certainty.
C. The corresponding peaks in the partialmolar heat capacity function were fitted by a four-state model, describing three well-defined unfoldingtransitions. Using CD spectroscopy, these are ascribed to the disruption of the domain interactions andsubsequent sequential unfolding of the two domains. The isolated N-terminal domain unfolds reversiblybetween pH 3.0 and pH 4.0 to >90% and at pH 7.0 to about 70%. In contrast, the isolated engineeredC-terminal domain only shows reversible thermal denaturation between pH 3.0 and pH 3.5. Neither N1/2nor C1/2 obeys the simple two-state mechanism of unfolding. Instead, both unfold via a partially structuredintermediate. In the case of N1/2, the intermediate exhibits native secondary structure and perturbed tertiarystructure, whereas for C1/2 the intermediate could not be defined with certainty.