Structural and ligand-binding properties of fatty acid-binding proteins (pFABP4 and pFABP5) in the gentoo penguin

Knowledge of the protein structure is considered a precondition for understanding their function. Crystallography remains the most powerful tool in that context, but crystal structure determination of macromolecules is often hampered by a lack of crystals suitable for diffraction. In our work, we have studied the crystallization of human serum albumin (HSA) and bovine beta-lactoglobulin (BLG) in the presence of yttrium chloride (and other multivalent salts) to yield high quality crystals with new crystal packing. We demonstrate that trivalent salts can be used to optimize crystallization conditions for globular proteins along general physical insights. We have revealed that negatively charged proteins at neutral pH in the presence of trivalent salts undergo a “reentrant condensation” phase behavior [1-3], i.e. a phase-separated regime occurs in between two critical salt concentrations, c* < c**, giving rise to a metastable liquid-liquid phase separation (LLPS) [3,4]. Crystallization from the condensed regime follows different mechanisms. Near c*, crystals grow following a classic nucleation and growth mechanism; near LLPS binodal, crystallization follows a two-step mechanism, i.e, crystals growth follows a metastable LLPS [4-8]. The yttrium ions are not only used to engineer crystallization, but are an integral part of the crystal lattice and can therefore be used to solve the phase problem using anomalous dispersion methods. The structure analysis demonstrates the specific binding of yttrium ions to surface-exposed glutamate and aspartate side chains contributed by different molecules in the crystal lattice. By bridging molecules in this manner, contacts between molecules are formed that enable the formation of a stable crystal lattice. Based on this bridging effect, an ion-activated patchy colloidal model was proposed to rationalize the phase behaviour observed experimentally [9]. References [1] F. Zhang et al., Phys. Rev. Lett. 2008, 101, 148101 [2] F. Zhang et al., Proteins 2010, 78, 3450 [3] F. Zhang et al., Soft Matter 2012, 8, 1313 [4] F. Zhang et al., J. Appl. Cryst. 2011, 44, 755 [5] F. Zhang et al., Faraday Discuss. 2012, 159, 313 [6] F. Roosen-Runge et al., J. Phys. Chem.B 2013, 117, 5777 [7] A. Sauter, et al. JACS, 2015, 137, 1485 [8] A. Sauter, et al. Faraday Discuss. 179, 41 [9] F. Roosen-Runge et al. Scientific Reports, 2014, 4, 7016.