Polymorphic Protein Crystal Growth: Influence of Hydration and Ions in Glucose Isomerase

Abstract

Crystal polymorphs of glucose isomerase were examined to characterize the properties and to quantify the energetics of protein crystal growth. Transitions of polymorph stability were measured in poly(ethylene glycol)/NaCl solutions, and one transition point was singled out for more detailed quantitative analysis. Single crystal x-ray diffraction was used to confirm space groups and identify complementary crystal structures. Crystal polymorph stability was found to depend on the NaCl concentration, with stability transitions requiring > 1 M NaCl combined with a low concentration of PEG. Both salting-in and salting-out behavior was observed and was found to differ for the two polymorphs. For NaCl concentrations above the observed polymorph transition, the increase in solubility of the less stable polymorph together with an increase in the osmotic second virial coefficient suggests that changes in protein hydration upon addition of salt may explain the experimental trends. A combination of atomistic and continuum models was employed to dissect this behavior. Molecular dynamics simulations of the solvent environment were interpreted using quasi-chemical theory to understand changes in protein hydration as a function of NaCl concentration. The results suggest that protein surface hydration and Na⁺ binding may introduce steric barriers to contact formation, resulting in polymorph selection.

Figure 1

Example of polyhedral and rectangular crystals in 4% PEG 10 kDa, 1.5 M NaCl, 10 mM MES, 5 mg mL^–1 GI pH 6.5 at 22 ± 2°C. Polyhedral crystals dissolve while the edges of rectangular crystals sharpen, indicating rectangle stability. Scale bar represents 250 μm.

Figure 2

Equilibrium solubility lines of rectangular (□) and polyhedral (○) polymorphs measured independently. The crystal type with the lower solubility at each [NaCl] is the more stable form. Error bars are from duplicate measurements. Lines are meant to guide the eye.

Figure 3

Normalized osmotic second virial coefficients measured by SIC. All solutions contained 4% (w/v) PEG 10 kDa in 10 mM MES at pH 6.5. Error bars are based on triplicate measurements. The solid line is a guide to the eye, and the dotted line indicates the hard-sphere interactions.

Figure 4

Calculated short-range interaction free energies between two GI molecules at crystal contacts using only the non-hydrogen protein atoms. Gap distance represents the distance relative to the crystallographic position along a line connecting the protein centers. The polyhedral crystal (7456) interaction energy is shown in blue. The two rectangular crystal interaction energies are shown in green and red for the 4565 and 2666 crystal contacts, respectively.

Figure 5

Calculated short-range interaction free energies between two GI molecules at a rectangular crystal contact (2666). Gap distance represents the distance relative to the crystallo-graphic position along a line connecting the protein centers. Solid red: interaction without water; solid blue: interaction including all crystallographic waters; dashed blue: interaction between fragments with bound water determined using MD for the contact pair; dashed red: interaction between fragments with bound water determined using MD for each isolated fragment.

Figure 6

Polyhedral crystal fragment 1 with bound water gained when the NaCl concentration was increased from 0 to 2 M. The side chain of aspartate 57 is shown with a bound Na⁺ ion (yellow) and gained water molecules (red).

Figure 7

Van der Waals surface of a GI tetramer. Colored residues are Asp, Glu and the C-terminus on each monomer, determined by MD simulations to bind Na⁺ ions and add water upon addition of 1 M (red) or 2 M (blue) NaCl. Asp 80, colored yellow, is where Na⁺ ions bind and add water at 2 M NaCl and is identified as a residue involved in salt-bridging within the polyhedral crystal contact.