C-13 NMR, X-RAY, AND DIFFERENTIAL SCANNING CALORIMETRY INVESTIGATIONSOF TRUNCATED BPTI (APROTININ) ANALOGS

Truncated BPTI missing residues 1 and 2 is investigated together with variants thereof (Lys-15, Arg-17, and Arg-42 are replaced by other res idues in various combinations). A comparison of the X-ray structure of BPTI with that of 3-58BPTI(K15R,R17A,R42S) shows only minor variation s 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 on e end. Comparisons of amide exchange rates show a dramatic increase fo r the most slowly exchanging NH protons of 3-58BPTI and the analogues thereof, as compared to those of the wildtype despite only small diffe rences in the structures, The amide exchange rates for truncated analo gues increase with decreasing TTEP (temperature top endothermic peak) values. On the basis of the known structural changes comparisons to C- 13 chemical shifts are made. C-13 chemical shifts are assigned using t he D-isotope and HMBC techniques. Excellent resolution is obtained in these 1D natural abundance spectra. C-13 NMR chemical shifts are shown to be able to gauge structural changes. A comparison of C-13 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 C-13 chemical shif ts are calculated. The difference in the calculated ring current effec ts are small comparing the wild-type with 3-58BPTI(K15R,R17A,R42S) pro vided the structures are relaxed. Protein unfolding as a function of p H and temperature is studied by DSC. Unfolding occurs at lower tempera ture with N-terminally truncated analogues, and the maximum is shifted toward higher pH.