Protein self-association in solution: the bovine pancreatic trypsin inhibitor decamer

Abstract

We have used magnetic relaxation dispersion to study bovine pancreatic trypsin inhibitor (BPTI) self-association as a function of pH, salt type and concentration, and temperature. The magnetic relaxation dispersion method sensitively detects stable oligomers without being affected by other interactions. We find that BPTI decamers form cooperatively under a wide range of solution conditions with no sign of dimers or other small oligomers. Decamer formation is opposed by electrostatic repulsion among numerous cationic residues confined within a narrow channel. Accordingly, the decamer population increases with increasing pH, as cationic residues are deprotonated, and with increasing salt concentration. The salt effect cannot be described in terms of Debye screening, but involves the ion-specific sequestering of anions within the narrow channel. The lifetime of the BPTI decamer is 101 +/- 4 min at 27 degrees C. We propose that the BPTI decamer, with a heparin chain threading the decamer channel, plays a functional role in the mast cell. We also detect a higher oligomer that appears to be a subcritical nucleation cluster of 3-5 decamers. We argue that monomeric crystals form at high pH despite a high decamer population in solution, because the ion pairs that provide the critical decamer-decamer contacts are disrupted at high pH.

FIGURE 1

¹H relaxation dispersion profile at 27°C from an aqueous solution containing 14.5 mM BPTI and 0.90 M NaCl at pH 4.5. The dispersion curve resulted from a constrained 3-Lorentzian fit according to Eqs. 1–4. The individual Lorentzian components (dashed curves) and the bulk water contribution (horizontal line) are also shown.

FIGURE 2

Dependence of the summed mean-square dispersion amplitude B on (a) salt concentration at pH 4.5, and (b) pH. Panel a shows results for NaCl at 27°C (solid circles) or 4°C (open circles), CsCl (open squares), and, from left to right, KSCN, Na₂SO₄, and NaI (solid squares). Panel b shows results for no salt or 0.10 M NaCl (solid circles), 0.50 or 0.70 M NaCl (open circles), and 0.10 M K₂HPO₄ (solid square).

FIGURE 3

¹H relaxation dispersion profiles from aqueous BPTI solutions at 27°C, pH 4.5 and the indicated NaCl concentrations. The data have been normalized to 14.5 mM BPTI. The dispersion curves resulted from constrained fits according to Eqs. 1–4 with parameter values as given in Table 1. All data in Figs. 3, 5, and 8 were fitted jointly with a common τ_R3.

FIGURE 4

Fraction BPTI oligomers as a function of (a) salt concentration at pH 4.5 and (b) nominal net structural charge of BPTI. The shaded region is bounded from below (open symbols) by the polymer fraction x₃ and from above (solid symbols) by the sum of the decamer and polymer fractions, x₂ + x₃ = 1 − x₁. Panel a shows results for NaCl at 27°C (circles) or 4°C (squares), and for CsCl (triangles). Panel b shows results for ≤0.1 M NaCl (circles) and for 0.1 M K₂HPO₄ (squares). Sample pH was converted to BPTI charge Z with the aid of the titration curve in Fig. 7. The actual net charge per BPTI molecule in the decamer may differ due to association-induced pK_ashifts.

FIGURE 5

¹H relaxation dispersion profiles from aqueous BPTI solutions at 27°C, pH 4.5 and the indicated NaCl or CsCl concentrations. The data have been normalized to 14.5 mM BPTI. The dispersion curves resulted from constrained fits according to Eqs. 1–4 with parameter values as given in Table 1. All data in Figs. 3, 5, and 8 were fitted jointly with a common τ_R3.

FIGURE 6

¹H relaxation dispersion profiles from aqueous BPTI solutions at 27°C, pH 4.5 and the indicated salt types and concentrations. The data have been normalized to 14.5 mM BPTI. The dispersion curves resulted from constrained fits according to Eqs. 1–4 with parameter values as given in Table 1. All data in Fig. 6 were fitted jointly with a common τ_R3.

FIGURE 7

Net charge of monomeric BPTI as a function of pH, calculated from published pK_a values (Wüthrich and Wagner, 1979). The pH values of samples examined here are indicated by points.

FIGURE 8

¹H relaxation dispersion profiles from aqueous BPTI solutions with 0.70 M NaCl at 27°C and pH 4.5 (Z = +6.4) or pH 2.5 (Z = +10.1). The data have been normalized to 14.5 mM BPTI. The dispersion curves resulted from constrained fits according to Eqs. 1–4 with parameter values as given in Table 1. All data in Figs. 3, 5, and 8 were fitted jointly with a common τ_R3.

FIGURE 9

¹H relaxation dispersion profiles from aqueous BPTI solutions at 27°C with no salt or 0.50 M NaCl and at pH 4.5 (Z = +6.4) or pH 12.2 (Z = −6.5). The data have been normalized to 14.5 mM BPTI. The dispersion curves resulted from constrained fits according to Eqs. 1–4 with parameter values as given in Table 1. The pH 12.2 data in Fig. 9 and the data in Fig. 6 were fitted jointly with a common τ_R3.

FIGURE 10

¹H relaxation dispersion profiles from aqueous BPTI solutions at 4°C and pH 4.5 with no salt or 0.66 M NaCl. The data have been normalized to 14.5 mM BPTI. The dispersion curves resulted from constrained fits according to Eqs. 1–4 with parameter values as given in Table 1.

FIGURE 11

The insert shows ¹H relaxation dispersion profiles recorded at 27°C from a 14.0 mM BPTI solution with 0.45 M NaCl and pH 4.5, made by mixing equal volumes of two BPTI solutions with no salt and 0.90 M NaCl. The initial dispersion profile is the average of the (concentration normalized) profiles from the original solutions and thus corresponds to the initial nonequilibrium state obtained by suddenly reducing the NaCl concentration from 0.90 to 0.45 M. The final dispersion profile was recorded one week after the salt jump. The main figure shows the time evolution of R₁, measured at 100 kHz (see dashed line in the insert). Note the change of scale at 300 min on the time axis. The curve resulted from a three-parameter fit according to Eq. 6. Omission of the t = 0 point, taken from the initial dispersion profile in the insert, had no discernable effect on the fit.

FIGURE 12

Standard free energy of association for BPTI decamers deduced from decamer populations determined by MRD at 27°C (circles, this work) or by SAXS at 20°C (squares, Hamiaux et al., 2000), both at pH 4.5. The fitted quadratic polynomial lacks physical significance. Error bars are propagated from an assumed uncertainty of ±0.03 in all decamer fractions.

FIGURE 13

The BPTI decamer, rendered with GRASP (Nicholls et al., 1991) using the atomic coordinates from the form A crystal structure 1BHC (Hamiaux et al., 1999). The upper pentamer contains monomers A–E, with E (left) and C (right) in front, and D removed. The lower pentamer contains monomers F–J, with H (left) and J (right) in front, and I removed. The color coding represents surface electrostatic potential on a scale from −15 (red) to +15 k_BT/e (blue), calculated with dielectric constants of 4 (protein) and 80 (solvent), 0.5 M salt, and full structural charges (Z = +6). Some of the Lys-46 residues (deep blue) can be seen protruding toward the center of the channel, whereas four Asp-3 residues (light red) are visible as protrusions near the equator at the extreme left and right of the decamer.

FIGURE 14

Relation between the polymer and decamer fractions at different salt concentrations (0.6–0.9 M NaCl, pH 4.5, with C_P = 14.5 mM, 27°C). The curve resulted from a fit according to Eq. 16, yielding M = 3.4.