Rostislaw Kaischew Institute of Physical Chemistry , Bulgarian Academy of Sciences , Acad. G. Bonchev Str. Bl. 11 , Sofia 1113 , Bulgaria.
Structural and Supramolecular Chemistry Laboratory, Institute of Nanoscience and Nanotechnology , National Centre for Scientific Research "Demokritos" , Athens 15310 , Greece.
ACS Appl Mater Interfaces. 2019 Apr 3;11(13):12931-12940. doi: 10.1021/acsami.8b20995. Epub 2019 Mar 22.
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.
大分子结晶对于包括结构生物学、药物设计、配方和输送、生物材料制造以及食品制备在内的许多科学领域都至关重要。本研究旨在通过理论和实验研究，探讨疏水性界面辅助蛋白质结晶，从而实现对结晶的控制。疏水性液体作为成核促进剂或抑制剂的应用很少被研究，为蛋白质结晶提供了一条未被充分探索的途径。从理论上讲，晶体成核被认为是一个两步过程，第一步是由于蛋白质在疏水性表面上的吸附而导致蛋白质浓度局部增加。随后，蛋白质在晶格中有序排列。通过计算维持二维核完整性的内聚能与倾向于破坏晶体的破坏能之和之间的平衡，可以接近水/疏水性物质界面上晶体成核的能量方面。这是通过比较形成晶体的单元共享的键数和指向溶液的晶体表面上未共享（悬垂）键数来实现的。同样的方法被扩展到水/疏水性液体界面上的三维蛋白质晶体成核。在实验方面，我们研究了蛋白质在油和其他疏水性液体（石蜡油、FC-70 氟利昂油和三种氯化碳氢化合物）上的结晶。比较了α-乳白蛋白和溶菌酶的结晶，并通过研究α-壳质蛋白、胰蛋白酶、胰岛素类似物和蛋白 Lpg2936 获得了额外的信息。根据蛋白质类型、浓度和界面老化时间，蛋白质在不同疏水性液体存在下表现出不同的结晶倾向。一些疏水性液体引起有效过饱和度的增加，这转化为与结晶溶液界面处晶体成核的增强，从而形成晶体。
