Berndt K D, Beunink J, Schröder W, Wüthrich K
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
Biochemistry. 1993 May 4;32(17):4564-70. doi: 10.1021/bi00068a012.
The three-dimensional structure of the basic pancreatic trypsin inhibitor (BPTI) contains four internal water molecules, which form a total of nine intermolecular hydrogen bonds with the BPTI polypeptide chain. To investigate the effect of such internal hydration on protein structure and stability, we displaced one of the internal water molecules in a recombinant BPTI analogue, BPTI(G36S), in which Gly 36 is replaced by serine. The replacement of a water molecule by the seryl side chain was established by the absence of the protein-water nuclear Overhauser effects (NOE) that had been attributed to the water molecule near Gly 36 in wild-type BPTI and by the presence of new, intramolecular NOEs to the hydroxyl proton of Ser 36. BPTI(G36S) has slightly reduced thermal stability compared to BPTI, corresponding to a destabilization by delta (delta G) approximately 0.7 kcal/M in 6 M guanidinium hydrochloride solution. Additionally, the stabilities of the complexes formed between BPTI(G36S) and trypsin, plasmin, or kallikrein are significantly reduced when compared to the corresponding complexes with wild-type BPTI.
基础胰蛋白酶抑制剂（BPTI）的三维结构包含四个内部水分子，这些水分子与BPTI多肽链总共形成九个分子间氢键。为了研究这种内部水合作用对蛋白质结构和稳定性的影响，我们在重组BPTI类似物BPTI(G36S)中置换了其中一个内部水分子，在该类似物中，甘氨酸36被丝氨酸取代。通过缺乏归因于野生型BPTI中甘氨酸36附近水分子的蛋白质 - 水核Overhauser效应（NOE）以及出现与丝氨酸36羟基质子的新的分子内NOE，确定了丝氨酰侧链取代了一个水分子。与BPTI相比，BPTI(G36S)的热稳定性略有降低，在6 M盐酸胍溶液中相当于约0.7千卡/摩尔的去稳定化（ΔΔG）。此外，与野生型BPTI形成的相应复合物相比，BPTI(G36S)与胰蛋白酶、纤溶酶或激肽释放酶形成的复合物的稳定性显著降低。
