Local fluctuations and global unfolding of partially folded BPTI detected by NMR
Abstract
The protein [14-38](Abu) is a chemically synthesized variant of bovine pancreatic trypsin inhibitor (BPTI) with the 14-38 disulfide bond intact and cysteines 5, 30, 51, and 55 replaced by α-amino-n-butyric acid (Abu). At 1-6°C and pH 4.5-6.5, [14-38](Abu) is partially folded with a native-like core [I]. Heteronuclear NMR spectra contain two, and in a few cases three or four, exchange cross peaks for each 15N-bound 1H, reporting the presence of two or more conformations that interconvert on a time scale of ≤ milliseconds. Thermodynamic analysis of 15N-1H exchange peak volumes as a function of temperature in the range 1-35°C indicates that partially folded [14-38](Abu) undergoes local segmental motions as well as cooperative global unfolding. The relative abundance of more folded versus disordered conformations changes throughout the molecule, indicating that various regions of the partially folded protein are disordered to different extents prior to onset of thermal denaturation. This system is unique in providing a measure of the populations of interconverting partially folded conformations, as well as a microscopic view of cooperative folding of a fluctuating ensemble. Although global thermal denaturation is cooperative, significant deviation from simple two-state behavior is reflected in several parameters, including the difference in T(m) for thermal unfolding measured by NMR versus circular dichroism.