Towards a molecular understanding of phase separation in the lens: a comparison of the X-ray structures of two high Tc gamma-crystallins, gammaE and gammaF, with two low Tc gamma-crystallins, gammaB and gammaD

gamma-Crystallins, although closely related in sequence, show intriguing differences in their temperature-dependent interactions: those that have a high or intermediate Tc for phase separation are cryoproteins whereas low Tc gamma-crystallins are not. To address the molecular basis of phase separation, X-ray crystallography has been used to define the structural differences between high and low Tc gamma-crystallins. A pre-requisite for this study was to clarify the assignment of bovine gene sequences to bovine gamma-crystallin proteins used for biophysical measurements. Based on nucleotide sequence analyses of gamma E and gamma F bovine crystallin genes, gamma F corresponds to the previously crystallised high Tc protein bovine gamma IVa and gamma E corresponds to the high Tc bovine protein fraction previously known as gamma IIIa. The gamma F sequence has enabled the completion of the refinement of the bovine gamma F crystal structure which shows that the molecule has an additional surface tryptophan explaining why gamma F has different spectroscopic properties from gamma B. A high Tc protein from rat lens, gamma E crystallin, has been crystallised and the X-ray structure solved at 2.3 A resolution. Comparison of the X-ray structures of two high Tc proteins, rat gamma E and bovine gamma F, with the structures of two low Tc proteins, bovine gamma B and bovine gamma D, shows that the main conformational change between high and low Tc proteins is in the cd surface loop of motif 3. All four structures have numerous ion pairs on their surfaces leading to a high surface charge density, yet with low overall charge. Comparison of the lattice contacts of the two high Tc proteins with the two low Tc gamma-crystallins indicates that these high Tc proteins utilise more amino-aromatic interactions such as between histidine and arginine. Comparison of the sequences of all the gamma-crystallins which have been characterised for phase separation temperature indicates that only residue Arg/Lys 163 uniquely distinguishes cryo from non-cryo gamma-crystallins and it is close to the altered surface loop. Although this region probably contributes to phase separation, Tc is likely to be a function of an overall global property that is responsive to overall charge distribution. Calculated dipole moments of native gamma-crystallins, low Tc gamma-crystallin sequences threaded into high Tc gamma-crystallin structures, and vice versa, show how both sequence and 3D structure contribute to this overall property. High Tc gamma-crystallins have on average higher Arg/Lys ratios and higher histidine content. It is hypothesised that this increases the proportion of surface static paired charged networks which thus reduces the repulsive hydration force and so increases the attractive interactions of the protein-rich phase in binary liquid phase separation.