High-throughput screening of a wide range of different conditions is typically required to obtain X-ray quality crystals of proteins for structure-function studies. The outcomes of individual experiments, i.e. the formation of gels, precipitates, microcrystals, or crystals, guide the search for and optimization of conditions resulting in X-ray diffraction quality crystals. Unfortunately, the protein will remain soluble in a large fraction of the experiments. In this paper, an evaporation-based crystallization platform is reported in which droplets containing protein and precipitant are gradually concentrated through evaporation of solvent until the solvent is completely evaporated. A phase transition is thus ensured for each individual crystallization compartment; hence the number of experiments and the amount of precious protein needed to identify suitable crystallization conditions is reduced. The evaporation-based method also allows for rapid screening of different rates of supersaturation, a parameter known to be important for optimization of crystal growth and quality. The successful implementation of this evaporation-based crystallization platform for identification and especially optimization of crystallization conditions is demonstrated using the model proteins of lysozyme and thaumatin.
SUMMARY
The eye lenses of the Antarctic nototheniid fishes that inhabit the perennially freezing Antarctic seawater are transparent at –2°C,whereas the cold-sensitive mammalian and tropical fish lenses display cold-induced cataract at 20°C and 7°C, respectively. No cold-cataract occurs in the giant Antarctic toothfish Dissostichus mawsoni lens when cooled to temperatures as low as –12°C, indicating highly cold-stable lens proteins. To investigate this cold stability, we characterised the lens crystallin proteins of the Antarctic toothfish, in parallel with those of the sub-tropical bigeye tuna Thunnus obesusand the endothermic cow Bos taurus, representing three disparate thermal climes (–2°C, 18°C and 37°C, respectively). Sizing chromatography resolved their lens crystallins into three groups,α/βH, β and γ, with γ crystallins being the most abundant (>40%) lens proteins in fish, in contrast to the cow lens where they comprise only 19%. The upper thermal stability of these crystallin components correlated with the body temperature of the species. In vitro chaperone assays showed that fish α crystallin can protect same-species γ crystallins from heat denaturation, as well as lysozyme from DTT-induced unfolding, and therefore are small Heat Shock Proteins (sHSP)like their mammalian counterparts. Dynamic light scattering measured an increase in size of αγ crystallin mixtures upon heating, which supports formation of the αγ complex as an integral part of the chaperone process. Surprisingly, in cross-species chaperone assays, tunaα crystallins only partly protected toothfish γ crystallins, while cow α crystallins completely failed to protect, indicating partial and no αγ interaction, respectively. Toothfish γ was likely to be the component that failed to interact, as the supernatant from a cowα plus toothfish γ incubation could chaperone cow γcrystallins in a subsequent heat incubation, indicating the presence of uncomplexed cow α. This suggests that the inability of toothfish γcrystallins to fully complex with tuna α, and not at all with the cowα crystallins, may have its basis in adaptive changes in the protein that relate to the extreme cold-stability of the toothfish lens.
Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a Mn(II)-binding site was designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215-222, 1997; Gengenbach et al. in Biochemistry 38:11425-11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k (cat)/K (M)) than the variant in the original design, mostly due to a stronger K (M) of 1.9 mM (vs. 4.1 mM). High-resolution X-ray crystal structures of a metal-free form and a form with Co(II) at the designed Mn(II) site were also obtained. The metal ion in the engineered metal-binding site overlays well with Mn(II) bound in MnP, suggesting that this variant is the closest structural model of the Mn(II)-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the metal; the metal-ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at the designed site, difference in the identity of metal ions (Co(II) rather than Mn(II)) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.