DeKoster G T, Robertson A D
Department of Biochemistry, The University of Iowa, Iowa City 52242, USA.
Biochemistry. 1997 Feb 25;36(8):2323-31. doi: 10.1021/bi962580b.
A synthetic gene for chicken ovomucoid first domain (OMCHI1) has been overexpressed in Escherichia coli. The resulting recombinant protein, rOMCHI1, is expressed and correctly folded without the use of fusion proteins or export secretion signal peptides incorporated into the gene. The thermostability of rOMCHI1 has been compared to that of the naturally occurring glycosylated OMCHI1 (gOMCHI1). The results of differential scanning calorimetry (DSC) studies show that the heat capacity change for unfolding, deltaCp, for both rOMCHI1 and gOMCHI1 is approximately 600 cal/(mol x K). At any given pH, however, the presence of N-linked carbohydrate increases the Tm for thermal unfolding of gOMCHI1 over rOMCHI1 by 2-4 degrees C, without changing the enthalpy of unfolding, delta H(degree)m. This suggests that the increased thermal stability of gOMCHI1 is entropic. Comparison of the unfolding thermodynamics of rOMCHI1 with those of turkey ovomucoid third domain (OMTKY3), which is 36% identical to rOMCHI1, reveals similar deltaCp values for both proteins, about 600 cal/(mol x K), but a reduction in delta H(degree)m of about 5 kcal/mol for rOMCHI1 at all temperatures. Decreases in delta H(degree)m for rOMCHI1 versus OMTKY3 may be explained by an overall less ordered native state in rOMCHI1. In the absence of a native structure for OMCHI1, the change in accessible surface area upon unfolding, deltaASA, was calculated using unfolding parameters 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 calculations suggest that the larger protein rOMCHI1 (Mr 7500) exposes less surface area than OMTKY3 (Mr 6100) upon thermal denaturation. Overall, structural energetic relationships may provide a useful framework for interpretation and comparison of thermodynamic data for structurally homologous proteins.
鸡卵类粘蛋白第一结构域（OMCHI1）的合成基因已在大肠杆菌中过表达。所产生的重组蛋白rOMCHI1得以表达且正确折叠，无需使用融合蛋白或基因中掺入的输出分泌信号肽。已将rOMCHI1的热稳定性与天然存在的糖基化OMCHI1（gOMCHI1）的热稳定性进行了比较。差示扫描量热法（DSC）研究结果表明，rOMCHI1和gOMCHI1展开时的热容变化ΔCp约为600 cal/（mol×K）。然而，在任何给定pH值下，N-连接碳水化合物的存在使gOMCHI1热展开的Tm比rOMCHI1高2 - 4℃，而不改变展开焓ΔH°m。这表明gOMCHI1热稳定性的提高是熵驱动的。将rOMCHI1与火鸡卵类粘蛋白第三结构域（OMTKY3）的展开热力学进行比较，OMTKY3与rOMCHI1有36%的序列同一性，结果显示两种蛋白质的ΔCp值相似，约为600 cal/（mol×K），但在所有温度下rOMCHI1的ΔH°m降低了约5 kcal/mol。rOMCHI1相对于OMTKY3的ΔH°m降低可能由rOMCHI1中整体无序程度较低的天然状态来解释。在没有OMCHI1天然结构的情况下，利用展开参数和结构能量关系计算了展开时可及表面积的变化ΔASA [Murphy & Freire (1992) Adv. Protein Chem. 43, 313 - 361; Murphy et al. (1993), Proteins: Struct., Funct., Genet. 15, 113 - 120]。这些计算表明，在热变性时，较大的蛋白质rOMCHI1（Mr 7500）比OMTKY3（Mr 6100）暴露的表面积更小。总体而言，结构能量关系可为解释和比较结构同源蛋白质的热力学数据提供一个有用的框架。
