Protein thermostabilization by proline substitutions in accordance with the proline rule

通过脯氨酸取代证实蛋白质的热稳定性-在Lys 457（β-转角的第二个位置）、Thr 440（环）和Ile 403（α-螺旋的N1位置）处用脯氨酸残基累积取代导致来自蜡状芽孢杆菌ATCC 7064的寡-1，6-葡萄糖苷酶的热稳定性的累加增强。β-转角的第2位和α-Herix的N1位增强效果最好。突变酶在结构和功能上与野生酶相似。这些观察结果与之前用9种突变酶获得的结果一致。寡聚-1，6-葡萄糖苷酶稳定性的评价-通过追踪不同浓度盐酸胍变性后的荧光强度，分析突变型和野生型寡聚-1，6-葡萄糖苷酶的可逆稳定性。然而，在用试剂变性的过程中未能检测到蛋白质的可逆性。突变蛋白和野生蛋白对盐酸胍具有不可逆的抗性，与之前检测到的热稳定性相一致。另一方面，来自热葡糖苷酶芽孢杆菌KP 1006的热稳定寡-1，6-葡糖苷酶（其在一级结构上与蜡状B酶具有72%的同一性）显示出对试剂的更不可逆抗性以及可逆稳定性。利用差示扫描量热法可以捕捉到蜡状B oligo-1，6-葡萄糖苷酶的可逆性。该系统揭示了每个野生型和突变型蛋白质的可逆稳定性与Tm值中的不可逆稳定性相关。

Confirmation of protein thermostabilization by proline substitutions---Cumulative replacements at Lys457 (the second position of beta-turn) , Thr440 (loop) and Ile403 (the N1 position of alpha-helix) with proline residues resulted in the additive enhancement in thermostability of oligo-1,6-glucosidase from Bacillus cereus ATCC7064. The enhancement was the most effective at the second position of beta-turn and at the N1 position of alpha-herix. The mutant enzymes showed similarities in structure and function as the wild one. These observations are consistent with the results obtained before with 9 mutant enzymes. Evaluation of stability for oligo-1,6-glucosidases---The reversible stability of the mutant and wild oligo-1,6-glucosidases was analyzed by tracing the intensities of their fluorescence after denatured with different concentrations of guanidine hydrochloride. However, the reversibility of the proteins failed to be detected during the denaturation with the reagent. The mutant and wild proteins showed the irreversible resistance to guanidine hydrochloride, corresponding to the thermal stability detected before. On the other hand, a thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006, which is 72%-identical with the B.cereus enzyme in primary structure, indicated more irreversible resistance to the reagent as well as reversible stability. The system of differential scanning calorimetry could catch the reversibility of B.cereus oligo-1,6-glucosidase. This system revealed that the reversible stability for each of the wild and mutant proteins was related to the irreversible one in Tm values.