Hansen P E, Zhang W, Lauritzen C, Bjørn S, Petersen L C, Norris K, Olsen O H, Betzel C
Department of Life Sciences and Chemistry, Roskilde University, Denmark.
Biochemistry. 1998 Mar 17;37(11):3645-53. doi: 10.1021/bi972282u.
Truncated BPTI missing residues 1 and 2 is investigated together with variants thereof (Lys-15, Arg-17, and Arg-42 are replaced by other residues in various combinations). A comparison of the X-ray structure of BPTI with that of 3-58BPTI(K15R,R17A,R42S) shows only minor variations for the backbone, but the lack of salt bridge between the terminals and the lack of two N-terminal residues provide a structure open at one end. Comparisons of amide exchange rates show a dramatic increase for the most slowly exchanging NH protons of 3-58BPTI and the analogues thereof, as compared to those of the wild-type despite only small differences in the structures. The amide exchange rates for truncated analogues increase with decreasing TTEP (temperature top endothermic peak) values. On the basis of the known structural changes comparisons to 13C chemical shifts are made. 13C chemical shifts are assigned using the D-isotope and HMBC techniques. Excellent resolution is obtained in these 1D natural abundance spectra. 13C NMR chemical shifts are shown to be able to gauge structural changes. A comparison of 13C chemical shifts of WT BPTI (aprotinin) and 3-58BPTI reveals effects caused by (i) the removal of the salt bridge of the terminii, (ii) the charge of the N-terminus, and (iii) the increased mobility of the side chain of Tyr-23. Small effects are also seen due to a conformational change of the aromatic ring of Phe-4. Ring current shifts at 13C chemical shifts are calculated. The difference in the calculated ring current effects are small comparing the wild-type with 3-58BPTI(K15R,R17A,R42S) provided the structures are relaxed. Protein unfolding as a function of pH and temperature is studied by DSC. Unfolding occurs at lower temperature with N-terminally truncated analogues, and the maximum is shifted toward higher pH.
研究了缺失第1和第2位残基的截短型BPTI及其变体（Lys-15、Arg-17和Arg-42以各种组合被其他残基取代）。将BPTI的X射线结构与3-58BPTI(K15R,R17A,R42S)的结构进行比较，结果表明主链仅有微小变化，但末端之间缺乏盐桥以及缺少两个N端残基使得结构在一端开放。酰胺交换率的比较表明，与野生型相比，3-58BPTI及其类似物中交换最慢的NH质子的交换率显著增加，尽管结构上只有微小差异。截短类似物的酰胺交换率随TTEP（温度最高吸热峰）值的降低而增加。基于已知的结构变化，对13C化学位移进行了比较。使用D-同位素和HMBC技术对13C化学位移进行了归属。在这些一维天然丰度谱中获得了出色的分辨率。结果表明13C NMR化学位移能够测量结构变化。对野生型BPTI（抑肽酶）和3-58BPTI的13C化学位移进行比较，揭示了由以下因素引起的影响：(i) 末端盐桥的去除；(ii) N端的电荷；(iii) Tyr-23侧链流动性的增加。由于Phe-4芳香环的构象变化也观察到了微小影响。计算了13C化学位移处的环电流位移。如果结构是松弛的，将野生型与3-58BPTI(K15R,R17A,R42S)的计算环电流效应差异较小。通过差示扫描量热法研究了蛋白质随pH和温度的展开情况。N端截短的类似物在较低温度下发生展开，且最大值向更高pH移动。
