The solution structure of bovine pancreatic trypsin inhibitor at high pressure

Abstract

The solution structure of bovine pancreatic trypsin inhibitor (BPTI) at a pressure of 2 kbar is presented. The structure was calculated as a change from an energy-minimized low-pressure structure, using (1)H chemical shifts as restraints. The structure has changed by 0.24 A RMS, and has almost unchanged volume. The largest changes as a result of pressure are in the loop 10-16, which contains the active site of BPTI, and residues 38-42, which are adjacent to buried water molecules. Hydrogen bonds are compressed by 0.029 +/- 0.117 A, with the longer hydrogen bonds, including those to internal buried water molecules, being compressed more. The hydrophobic core is also compressed, largely from reduction of packing defects. The parts of the structure that have the greatest change are close to buried water molecules, thus highlighting the importance of water molecules as the nucleation sites for volume fluctuation of proteins in native conditions.

Figure 1.

Radial distribution functions for low- and high-pressure BPTI structures. The family 8 results are shown as an illustration. Distances were calculated between all pairs of atoms (including hydrogens) and placed into 0.1 Å bins. The distributions were weighted by dividing the number in each bin by the square of the radius. (A) The functions for the low-pressure structure (solid line) and the high-pressure structure (dashed line). (B) The difference (high − low), such that a positive peak at any distance means that there are more atom pairs at this distance in the high-pressure structure than there are in the low-pressure structure.

Figure 2.

Difference distance matrices, for (A) family 8, (B) family 13, and (C) family 19. The contours are plotted at contour levels of ±0.6, ±0.5, ±0.4 Å, and indicate in red parts of the structure where distances are shorter and in blue parts where the distances are longer. The regular secondary structure elements of the structure are indicated.

Figure 3.

RMS distance between corresponding backbone atoms in low- and high-pressure structures for family 19 by residue, after superposition of backbone heavy atoms in regular secondary structures. The line at 0.35 Å indicates the cutoff used for Figure 4 ▶.

Figure 4.

Ribbon representation of BPTI (drawn using GRASP; Nicholls et al. 1991). Residues colored black are those for which the RMS distance between corresponding residues in low- and high-pressure structures is greater than 0.35 Å (Fig. 3 ▶). Named residues are those that show large changes in their relative distances to other parts of the protein (Fig. 2 ▶). The four buried water molecules are also indicated.

Figure 5.

Direction of movement of heavy atoms in BPTI with pressure. An angle of 0 corresponds to movement towards the center of mass. Random motion would give a horizontal distribution at n = 1. (Solid line) Family 8; (dashed line) family 13; (dotted line) family 19.

Figure 6.

Hydrogen bond lengths in low- and high-pressure structures. Hydrogen bonds were identified using the method of Kabsch and Sander (1983). (•) Helical structure; (▴) sheet structure; +, loop; (▪) hydrogen bond to buried water molecule. The line of best fit is indicated by a solid line, and the y = x diagonal by a dashed line.

Figure 7.

Changes in hydrogen bond length with pressure, showing locations on the structure of BPTI. Hydrogen bonds are indicated by thick colored lines, color-coded by the change in distance with pressure: dark blue, shorter by more than 0.1 Å; cyan, shorter by less than 0.1 Å; yellow, longer by more than 0.1 Å; green, longer by more than 0.1 Å. Figure produced using MOLSCRIPT (Kraulis 1991).

Figure 8.

Relative locations of Cys51 and Phe45 in low- and high-pressure structures. The two structures were superimposed on the ring atoms of Phe45.