X-Ray Crystallography: Protein Sample Requirements
A precondition for a successful crystallographic screening is a pure, stable, and monodisperse protein sample.
In the process of protein production, controls such as SDS-gels already indicate towards the purity of a protein sample. Additionally, during standard concentration determination via UV/VIS, contamination by DNA or RNA can be assessed. In the following section, typical methods for assessment of protein quality are described with the focus on crystallography-grade protein quality.
After protein production and before crystallization it is essential to check for protein quality. Initial assessment via native Mass-Spec can confirm the correct size of the purified protein with more precision than a SDS gel and can also give first indication regarding oligomerization states or very tightly bound cofactors as well as the presence of posttranslational modifications. Even if the purified protein has the correct molecular weight, it can still be unclear whether the protein is correctly folded.
Protein folding is a key process in biology since it is ultimately responsible for their biological function. Proteins that are misfolded or not folded at all may lead to unsatisfactory results in subsequent experiments or significant loss of protein material due to aggregation over time. Ultraviolet circular dichroism (CD) is the method of choice to monitor changes of protein structure in solution providing information of secondary protein structure, hence, the correct folding.
Protein samples need not only to be correctly folded but also stable and monodisperse. Thermal shift assay (TSA) measures the melting temperature of a protein (Tm), which is an indication of protein stability. Several factors such as pH, salt, cofactor, or buffer composition influence protein stability. Ideally, the protein is measured and stored in a buffer where it is more stable.
Melting curve of T. cruzi FPPS protein: the addition of Mg2+ increases protein stability by about 5°C (Francesca Magari)
Proteins also exhibit different stability against freeze-thaw cycles. This stability is a very important factor in inter-lab and general process optimization and can be improved via buffer optimization. Proteins that are not stable against freeze-thaw cycles have to be crystallized immediately after purification and cannot be sent on dry ice. The lifetime of such proteins can usually be extended via storage at 4°C or on wet ice but is much shorter than at -80°C. However, there are some rare cases in our experience, in which storage at lower temperatures is disfavored and the protein must be kept at room temperature or above.
Protein can be stable in a certain buffer, but the composition of the sample is not necessarily monodisperse. Monodispersity means that the protein exists in solution as a single oligomeric species, i.e., monomer or dimer, and is free of non-specific oligomers and aggregates. This can be checked with dynamic light scattering (DLS).
In summary, the quality of crystallography-grade protein material is significantly higher compared to assay-grade protein. A checklist for crystallography-grade protein samples are
- free from DNA/RNA
- stable: Tm > 30 °C
- properly folded
- resistant to freeze-thaw-cycles
- high purity according to analytics