Temperature-Dependent Interactions Explain Normal and Inverted Solubility in a γD-Crystallin Mutant

Abstract

performed experiments and analysed data. A.K., I.A. P.C. and J.J.M wrote the manuscript. AbstractProtein crystal production is a major bottleneck for the structural characterisation of proteins. To advance beyond large-scale screening, rational strategies for protein crystallization are crucial. Understanding how chemical anisotropy (or patchiness) of the protein surface due to the variety of amino acid side chains in contact with solvent, contributes to protein-protein contact formation in the crystal lattice is a major obstacle to predicting and optimising crystallization. The relative scarcity of sophisticated theoretical models that include sufficient detail to link collective behaviour, captured in protein phase diagrams, and molecular level details, determined from high-resolution structural information is a further barrier. Here we present two crystals structures for the P23T+R36S mutant of γD-crystallin, each with opposite solubility behaviour -one melts when heated, the other when cooled. When combined with the protein phase diagram and a tailored patchy particle model we show that a single temperature dependent interaction is sufficient to stabilise the inverted solubility crystal. This contact, at the P23T substitution site, relates to a genetic cataract and reveals at a molecular level, the origin of the lowered and retrograde solubility of the protein. Our results show that the approach employed here may present an alternative strategy for the rationalization of protein crystallization.

Using Schematic Models to Understand the Microscopic Basis for Inverted Solubility in γD-Crystallin

“…6ETC 30 ), and DBN (PDB ID: 6ETA 30 ). Of these, only WT, R36S, and R58H do not have a mutation at the 23 rd residue.…”

“…3.1), Ref. 30 proposed a reasonable correction would be to halve patch energies. For ε tot = 30, we have n * w ∼ 35 (Fig.…”

“…A number of modifications were made to the geometry of the actual protein crystal to be able to represent the proteins as spherical objects in an orthorhombic unit cell and are detailed in the SI of Ref. 30.…”

“…6ETC 30 ), and DBN (PDB ID: 6ETA within Chimera, 43 and all crystal water molecules are removed. In order to estimate the error on these measured hydrophobicities, 100 structures per crystal are created by perturbing each particle coordinate by a random number selected from a Gaussian distribution with standard deviation corresponding to the coordinate error specified in the PDB file.…”

Looped liquid-liquid coexistence in protein crystallization

“…However, understanding precisely how a protein solution becomes a protein crystal is both a fundamental challenge and an important question in biological materials design [2][3][4][5]. In computer models of crystallization, proteins are represented in terms of sticky, patchy spheres [6,7] or simple geometric objects [8,9] with short-ranged [10] and highly directional interactions [8,[11][12][13][14]. These models are described by a phase diagram that exhibits a fluid-solid transition and metastable liquid-liquid separation with an upper critical solution temperature (UCST).…”