LOCAL FLUCTUATIONS AND GLOBAL UNFOLDING OF PARTIALLY FOLDED BPTI DETECTED BY NMR

The protein [14-38](Abu) is a chemically synthesized variant of bovine pancreatic trypsin inhibitor (BPTI) with the 14-38 disulfide bond int act and cysteines 5, 30, 51, and 55 replaced by alpha-amino-n-butyric acid (Abu). At 1-6 degrees C and pH 4.5-6.5, [14-38](Abu) is partially folded with a native-like core [1]. Heteronuclear NMR spectra contain two, and in a few cases three or four, exchange cross peaks for each N-15-bound H-1, reporting the presence of two or more conformations th at interconvert on a time scale of greater than or equal to millisecon ds. Thermodynamic analysis of N-15-H-1 exchange peak volumes as a func tion of temperature in the range 1-35 degrees C indicates that partial ly folded [14-38](Abu) undergoes local segmental motions as well as co operative global unfolding. The relative abundance of more folded vers us disordered conformations changes throughout the molecule, indicatin g that various regions of the partially folded protein are disordered to different extents prior to onset of thermal denaturation. This syst em is unique in providing a measure of the populations of interconvert ing partially folded conformations, as well as a microscopic view of c ooperative folding of a fluctuating ensemble. Although global thermal denaturation is cooperative, significant deviation from simple two-sta te behavior is reflected in several parameters, including the differen ce in T-m for thermal unfolding measured by NMR versus circular dichro ism.