Attenuated total reflection-FT-IR spectroscopic imaging of protein crystallization

Protein crystallization is of strategic and commercial relevance in the post-genomic era because of its pivotal role in structural proteomics projects. Although protein structures are crucial for understanding the function of proteins and to the success of rational drug design and other biotechnology applications, obtaining high quality crystals is a major bottleneck to progress. The major means of obtaining crystals is by massive-scale screening of a target protein solution with numerous crystallizing agents. However, when crystals appear in these screens, one cannot easily know if they are crystals of protein, salt, or any other molecule that happens to be present in the trials. We present here a method based on Attenuated Total Reflection (ATR)-FT-IR imaging that reliably identifies protein crystals through a combination of chemical specificity and the visualizing capability of this approach, thus solving a major hurdle in protein crystallization. ATR-FT-IR imaging was successfully applied to study the crystallization of thaumatin and lysozyme in a high-throughput manner, simultaneously from six different solutions. This approach is fast as it studies protein crystallization in situ and provides an opportunity to examine many different samples under a range of conditions.

The relationship between charge density and polyelectrolyte brush profile using simultaneous neutron reflectivity and in situ attenuated total internal reflection FTIR

We report on a novel experimental study of a pH-responsive polyelectrolyte brush at the silicon/D2O interface. A poly[2-(diethylamino)ethyl methacrylate] brush was grown on a large silicon crystal which acted as both a substrate for a neutron reflectivity solid/liquid experiment but also as an FTIR-ATR spectroscopy crystal. This arrangement has allowed for both neutron reflectivities and FTIR spectroscopic information to be measured in parallel. The chosen polybase brush shows strong IR bands which can be assigned to the N-D+ stretch, D2O, and a carbonyl group. From such FTIR data, we are able to closely monitor the degree of protonation along the polymer chain as well as revealing information concerning the D2O concentration at the interface. The neutron reflectivity data allows us to determine the physical brush profile normal to the solid/liquid interface along with the corresponding degree of hydration. This combined approach makes it possible to quantify the charge on a polymer brush alongside the morphology adopted by the polymer chains. © 2013 American Chemical Society.