Integral membrane proteins (IMPs) represent a large class of targets for rational drug design, with approximately half of new drugs directly targeting IMPs. Crystallization and structure determination of IMPs are challenging, and currently <1% of human IMP structures have been solved. This difficulty arises from the inherent tendency of membrane proteins to reside in a two-dimensional (2D) lipid environment, whereas most conventional protein crystallization techniques require extraction and crystallization in a three-dimensional (3D) aqueous solution. Here, 2D IMP crystals were grown within living cells and second harmonic generation (SHG) microscopy was used to rapidly and non-destructively detect crystal formation. The presence of 2D crystals can be used as an indication of IMP targets likely to form larger 3D crystals within lipidic mesophases, or 2D crystals can be extracted from cells and used directly for structure determination using electron diffraction, termed �cellular crystallography.� For the figure shown below, H. halobium cells were grown under conditions known to lead to the formation of 2D crystalline patches of bacteriorhodopsin. SHG signal was observed in H. halobium cells, indicating the presence of 2D crystals. SHG results were subsequently confirmed by electron diffraction.

Detection of 2D integral membrane protein crystals within living cells using second harmonic generation microscopy

246th National Meeting (2013)

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