title: Reduced BPTI is collapsed. A pulsed field gradient NMR study of unfolded and partially folded bovine pancreatic trypsin inhibitor.
abstract: 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 alpha-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 degree -the diffusion coefficient extrapolated to infinite dilution-is determined. From D degree, a value of the hydrodynamic radius. Rh, is computed from the Stokes-Einstein relationship. At pH 4.5, [R]Abu has an Rh value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental Rh 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.
authors.affiliation: Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
authors.forename: H
authors.initials: H
authors.lastname: Pan
authors.forename: G
authors.initials: G
authors.lastname: Barany
authors.forename: C
authors.initials: C
authors.lastname: Woodward
mesh_terms: D007611:Aprotinin / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D007611:Aprotinin