Thermodynamics of Unfolding for Kazal-Type Serine Protease Inhibitors: Entropic Stabilization of Ovomucoid First Domain by Glycosylation
详细信息 查看全文
- 作者：Gregory T. DeKoster and and Andrew D. Robertson
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：February 25, 1997
- 年：1997
- 卷：36
- 期：8
- 页码：2323 - 2331
- 全文大小：254K
- 年卷期：v.36,no.8(February 25, 1997)
- ISSN：1520-4995
文摘
A synthetic gene for chicken ovomucoid first domain(OMCHI1) has been overexpressed inEscherichia coli. The resulting recombinant protein,rOMCHI1, is expressed and correctly folded withoutthe use of fusion proteins or export secretion signal peptidesincorporated into the gene. The thermostabilityof rOMCHI1 has been compared to that of the naturally occurringglycosylated OMCHI1 (gOMCHI1).The results of differential scanning calorimetry (DSC) studiesshow that the heat capacity change forunfolding, 
Cp, for both rOMCHI1and gOMCHI1 is approximately 600 cal/(mol·K). At any givenpH,however, the presence of N-linked carbohydrate increases theTm for thermal unfolding of gOMCHI1over rOMCHI1 by 2-4 
C, without changing the enthalpy of unfolding,Hm. This suggests thattheincreased thermal stability of gOMCHI1 is entropic. Comparison ofthe unfolding thermodynamics ofrOMCHI1 with those of turkey ovomucoid third domain (OMTKY3), which is36% identical to rOMCHI1,reveals similar Cp values for bothproteins, about 600 cal/(mol·K), but a reduction inHm of about 5kcal/mol for rOMCHI1 at all temperatures. Decreases inHm for rOMCHI1 versus OMTKY3 maybeexplained by an overall less ordered native state in rOMCHI1. Inthe absence of a native structure forOMCHI1, the change in accessible surface area upon unfolding, ASA,was calculated using unfoldingparameters and structural energetic relationships [Murphy & Freire(1992) Adv. Protein Chem. 43, 313-361; Murphy et al. (1993), Proteins: Struct., Funct.,Genet. 15, 113-120]. These calculationssuggestthat the larger protein rOMCHI1 (Mr 7500)exposes less surface area than OMTKY3 (Mr 6100)uponthermal denaturation. Overall, structural energetic relationshipsmay provide a useful framework forinterpretation and comparison of thermodynamic data for structurallyhomologous proteins.
Cp, for both rOMCHI1and gOMCHI1 is approximately 600 cal/(mol·K). At any givenpH,however, the presence of N-linked carbohydrate increases theTm for thermal unfolding of gOMCHI1over rOMCHI1 by 2-4 C, without changing the enthalpy of unfolding,Hm. This suggests thattheincreased thermal stability of gOMCHI1 is entropic. Comparison ofthe unfolding thermodynamics ofrOMCHI1 with those of turkey ovomucoid third domain (OMTKY3), which is36% identical to rOMCHI1,reveals similar Cp values for bothproteins, about 600 cal/(mol·K), but a reduction inHm of about 5kcal/mol for rOMCHI1 at all temperatures. Decreases inHm for rOMCHI1 versus OMTKY3 maybeexplained by an overall less ordered native state in rOMCHI1. Inthe absence of a native structure forOMCHI1, the change in accessible surface area upon unfolding, ASA,was calculated using unfoldingparameters and structural energetic relationships [Murphy & Freire(1992) Adv. Protein Chem. 43, 313-361; Murphy et al. (1993), Proteins: Struct., Funct.,Genet. 15, 113-120]. These calculationssuggestthat the larger protein rOMCHI1 (Mr 7500)exposes less surface area than OMTKY3 (Mr 6100)uponthermal denaturation. Overall, structural energetic relationshipsmay provide a useful framework forinterpretation and comparison of thermodynamic data for structurallyhomologous proteins.