Specific isotope labeling holds many advantages for NMR studies of macromolecular systems. We are developing Pattern Specific Aromatic Labeling (PSAL) to study ever larger systems. The distinct chemical shifts of phenylanine, tryosine, and tryptophan side chains can be implemented as initiators in saturation transfer experiments and are readily distinguished in inter- and intramolecular NOE experiments. Successful production of 1H-13C phenylalanine has been established and recombinant protein expression shows >95% incorporation. Optimization of pattern specifically labeled phenylalanine expression is underway as well as production of tryonsine and tryptophan. The goal of this project is to make a widely applicable, inexpensive set of reagents to explore macromolecular complexes and membrane proteins beyond current circumstances.
Pattern Specific Phenylalanine Labeling
Cα, Cγ, Cε pattern specifically labeled phenylalanine was produce (inset in left panel). Recombinant ubiquitin was expressed after supplementing with Cα, Cγ, Cε labeled phenylalanine. A broadband 13C-HSQC was collected from the purified protein, with only side chain 1Hε-13Cε and backbone 1Hα-13Cα resonances from the two phenylalanine residues in ubiquitin present (left). An aromatic selective version of the same experiment shows no carbon-carbon coupling (right). All data collected at 500 MHz.
Pattern Specifically Labeled Ubiquitin (coming soon)