Protein Detergent Complex (PDC) Identification
One of the most commonly encountered problems in membrane protein crystallization is the identification of detergents to form protein detergent complexes (PDC). Applying DLS in situ compensates some former obstacles of this method e.g. the required sample volume, cuvette cleaning and the limit to one sample at the time. In situ DLS makes the use of multi-well plates possible to allow a full scale screening of 96 conditions at a time. In combination with the use of very low quantities of sample (~500 nl or even less) and an automated way to perform measurements, DLS becomes high throughput capable. These are the prerequisites for a detergent screening in which a PDC can be identified by size distribution and size comparison. Since PDCs are uniform populations of structurally intact macromolecules they provide clearly distinguishable distribution signatures which are easy to identify among many other possible size distributions. The strategy to identify the right buffer conditions to form a PDC applying in situ DLS is based on size comparison of pure “empty” micelles with the protein solution containing the same detergent. Pure micelles are slightly smaller than PDCs. PDCs should show a dominant peak at the desired size of approximately 5 – 10 nm and stable over time. Assuming that the membrane protein is already dissolved in some kind of buffer this is the initial point to start the screening. The most convenient way to screen is by adding an excessing amount of the new detergent buffer to the sample. Since the DLS detection volume is 500 nl and the ratio of new buffer and protein sample is 4:1 only 100 nl sample are required for a single test. A PDC shows a significant peak which is usually slightly larger and broader as the peak from the pure detergent, since a mixture of “empty” micelles and PDCs is likely.
Protein aggregating and Protein Detergent Complex DLS Sigantures
Identification of Protein Detergent Complexes based upon solubilization in detergent micelles. To study such solubilization we applied in situ DLS.
Comparison of "emty" Micelles Size and PDC
An example for PDC Identification based on size comparison. Since a detergent micelle is a highly compact and stochiometrical uniform particle, all types of PDC's are larger. Besides the absolute size, uniformity and stability over time are indicators for PDC-formation.