Although nearly half of today's major pharmaceutical drugs target human integral membrane proteins (hIMPs), inefficiencies in protein production have historically represented a primary bottleneck for hIMP characterization. A combination of cell-free expression and combinatorial isotope-labeling strategy enabled us a highly time-efficient structure determination of hIMPs by solution NMR spectroscopy (Klammt et al., 2012, Nature Methods). With the speed of adding six new structures over 18 months time period to 30 hIMP structures that were available in the Protein Data Bank at the time of deposition, the strategy offers a significant improvement in efficiency of the production and structural characterization of hIMPs by solution NMR spectroscopic methods. Further improvement involves:
1) direct labeling via non-natural amino acids expressed and incorporated directly to the target hIMPs to bypass generating Cys-free mutants for PRE analysis, and;
2) accelerated Molecular Dynamics simulation to derive lipid membrane-embedded conformations of target structures post experimental determination by NMR spectroscopy.
With the addition of rigorous computational stragety including accelerated molecular dynamics, these efforts will accelerate not only structure elucidation of the target hIMPs as well as their related hIMP sequences in the protein sequence space, but also their detailed functional analyses ultimately aimed for drug design and discovery on the human targets.