Pulsed field gradient NMR was used to measure the hydrodynamic behavior of unfolded variants of bovine pancreatic trypsin inhibitor (BPTI). The unfolded BPTI species studied were [R](Abu), at pH 4.5 and pH 2.5, and unfolded [14-38](Abu), at pH 2.5. These were prepared by chemical synthesis [R](Abu) is a model for reduced BPTI; all cysteine residues are replaced by α-amino-n-butyric acid (Abu). [14-38](Abu) retains cysteines 14 and 38, which form a disulfide bond, while the other cysteine residues are replaced by Abu. In the PFG experiments, the diffusion coefficient is measured as a function of protein concentration, and the value of D°-the diffusion coefficient extrapolated to infinite dilution-is determined. From D°, a value of the hydrodynamic radius, R(h), is computed from the Stokes-Einstein relationship. At pH 4.5, [R](Abu) has an R(h) value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental R(h) value is the same as for a random coil. In view of the changes in NMR- detected structure of [R](Abu) at pH 4.5 versus pH 2.5 (Pan H, Barbar E, Barany G, Woodward C. 1995. Extensive non-random structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 34:13974-13981), the collapse of reduced BPTI at pH 4.5 may be associated with the formation of non-native hydrophobic clusters of pairs of side chains one to three amino acids apart in sequence. The diffusion constant of [14-38](Abu) was also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially folded [14-38](Abu), relative to native BPTI, is similar to the increase in radius of gyration measured for other proteins under 'molten globule' conditions.
- Bovine pancreatic trypsin inhibitor
- Protein folding
- Protein hydrodynamics
- Pulsed field gradient NMR
- Unfolded proteins