Technical Highlight - August 2012
Short description: Backbone structure determination of human integral membrane proteins is enabled by cell-free protein expression followed by solution NMR spectroscopy.
Integral membrane proteins (IMPs) sit at the interface of the cell and its outside world, and thus have key roles in transport, intercellular signaling and other processes. As such, they are targets for many pharmaceutical drugs available today. However, being transmembrane proteins, they are difficult to express, purify and crystallize, so little structural data are available to better understand and manipulate this important class of proteins.
Choe and colleagues have been developing protocols to overcome these roadblocks. Since the usual workhorse for protein overexpression, Escherichia coli, lacks the translocation machinery needed for human IMP (hIMP) production, they developed an E. coli-derived cell-free expression system. hIMPs produced in this system form precipitates, which can subsequently be solubilized with mild detergents. The researchers termed this “precipitating cell-free” (P-CF) expression. Another important advantage of this system is that proteins can be uniformly labeled by adding labeled amino acid combinations during synthesis, enabling combinatorial dual labeling (CDL). This CDL strategy facilitated resonance assignment, expediting this work.
For their initial analysis, the authors selected 15 hIMPs and expressed them in an optimized version of the P-CF system. Fourteen of these resulted in natively folded protein suitable for characterization without the need for purification. After obtaining [1H-15N] TROSY-HSQC spectra, N-H cross-peaks could be assigned for six of the proteins using the CDL strategy. The researchers then proceeded to calculate backbone structures for these, all of which now await functional characterization. Encouraged by these results, the group expanded their initial 15 targets by adding 135, and have spectra for an additional 32 hIMPs ready for backbone structure determination.
Solved in just 18 months, the initial six structures add significantly to the only 30 hIMP structures that were previously in the PDB. Based on these structures, the researchers calculated structural models for hundreds of proteins in UniProtKB with >30% sequence identity to these proteins. The plethora of new hIMP structural information should move the characterization and manipulation of these pharmaceutically important proteins forward.
C. Klammt et al. Facile backbone structure determination of human membrane proteins by NMR spectroscopy.
Nat. Methods (20 May 2012). doi:10.1038/nmeth.2033