Macromolecular
crystallization is crucial to a large number of
scientific fields, including structural biology; drug design, formulation,
and delivery; manufacture of biomaterials; and preparation of foodstuffs.
The purpose of this study is to facilitate control of crystallization,
by investigating hydrophobic interface-assisted protein crystallization
both theoretically and experimentally. The application of hydrophobic
liquids as nucleation promoters or suppressors has rarely been investigated,
and provides an underused avenue to explore in protein crystallization.
Theoretically, crystal nucleation is regarded as a two-step process,
the first step being a local increase in protein concentration due
to its adsorption on the hydrophobic surface. Subsequently, the protein
is ordered in a crystal lattice. The energetic aspect of crystal nucleation
on water/hydrophobic substance interfaces is approached by calculating
the balance between the cohesive energy maintaining integrity of the
two-dimensional crystal nucleus and the sum of destructive energies
tending to tear up the crystal. This is achieved by comparing the
number of bonds shared by the units forming the crystal and the number
of unshared (dangling) bonds on the crystal surface pointing toward
the solution. The same approach is extended to three-dimensional protein
crystal nucleation at water/hydrophobic liquid interfaces. Experimentally,
we studied protein crystallization over oils and other hydrophobic
liquids (paraffin oil, FC-70 Fluorinert fluorinated oil, and three
chlorinated hydrocarbons). Crystallizations of α-lactalbumin
and lysozyme are compared, and additional information is acquired
by studying α-crustacyanin, trypsin, an insulin analogue, and
protein Lpg2936. Depending on the protein type, concentration, and
the interface aging time, the proteins exhibit different crystallization
propensities depending on the hydrophobic liquid used. Some hydrophobic
liquids provoke an increase in the effective supersaturation, which
translates to enhancement of crystal nucleation at their interface
with the crystallization solution, leading to the formation of crystals.