Influence of pH on Two-Dimensional Streptavidin Crystals

Abstract

To obtain a general understanding of the effect of intermolecular interactions on the mechanisms of two-dimensional protein crystallization, we grow protein crystals and elicit a bulk molecular manipulation by changing system pH. Two-dimensional crystals of the bacterial protein streptavidin grown on a biotinylated lipid monolayer at an air-water interface, in the presence of the noncrystallizable impurity avidin, exhibit crystallographic and morphological changes as a function of subphase pH. Large twodimensional crystalline arrays form within minutes across a pH range from 1.5 to 11. Crystals exhibit different pH-dependent structures, lattices with P1 symmetry for 1.5 < pH < 5, P1 and P2 lattices for 5 < pH < 6, and C222 lattices for 7 < pH < 11. P1 crystals nucleate rapidly and form thin needle-shaped crystals consistent with a strong growth anisotropy between the two crystallographic growth directions. C222 crystals grow more isotropically and exhibit H-and X-shapes. The nucleation rates and aspect ratios of C222 crystals are also pH-dependent, both properties increasing with increasing pH. The transition from C222 to P1 or P2 crystals can be accomplished in minutes by lowering the system pH. The reverse transition, however, does not occur subsequent to a corresponding increase in system pH. Instead, new C222 crystals form, but no reconfiguration of existing crystals is observed.

Coupling between protein-laden films and a shearing bulk flow

“…We estimate the area coverage in cases where crystals are observed to be about 10%, however the signal is of the order of the variance of the measurement. In a quiescent system at large supersaturation, [32] gives the area fraction to be as high as 20% for a buffer with pH 4.3. At the shear rates used in our experiment, no deformations of the crystals were detected, and during the short time they are in the field of view of the microscope no rotation can be detected.…”

“…For the preparation of the buffer and the monolayer mixture, the procedures in [12,14,20] were followed with some modifications. The experiments were conducted using an aqueous buffer prepared with de-ionized water (Milli-Q filtered, with resistivity greater than 18 M cm).…”

Simulation of two‐dimensional streptavidin crystallization

“…2D protein islands may also serve as a model clarifying the lateral self‐organization of proteins on biological surfaces 3. In addition, 2D protein arrays are useful in protein structure determination in cases in which proteins are recalcitrant to 3D crystallization or in situations in which protein availability is scarce 4. They may also function as templates for epitaxial growth of 3D protein crystals and serve for an improved understanding of the mechanisms of protein crystallization in general.…”

“…One of the best examples in the field under consideration is 2D crystallization of streptavidin at a biotinylated lipid layer 5. In this case, the formation of large 2D islands (up to ∼100 μm) occurs4 within minutes across a pH range of 1.5–11. With increasing pH, the streptavidin structures varies from needle‐shaped crystals with P1 and/or P2 symmetry at 1.5 ≤ pH ≤ 5 to more isotropic X, H, or rectangular C222 islands at 7 ≤ pH ≤ 11.…”

“…With increasing pH, the streptavidin structures varies from needle‐shaped crystals with P1 and/or P2 symmetry at 1.5 ≤ pH ≤ 5 to more isotropic X, H, or rectangular C222 islands at 7 ≤ pH ≤ 11. Despite a large number of reports2, 4–10 about 2D streptavidin crystallization, the physics behind this phenomenon are still poorly understood. The kinetics of streptavidin crystallization have not been experimentally studied in detail either.…”