Optimized histidine-specific isotopic labeling in proteins overproduced in Escherichia coli

The use of specific isotope labeling methods has significantly enhanced structural and kinetic research in protein science using a variety of biophysical techniques. Histidine plays key roles in a wide range of protein functions: enzymatic catalysis, metal ion coordination, protein stability, and redox reactions. In this article, we report a method for optimized histidine-specific isotopic labeling in proteins overproduced in Escherichia coli. We developed and validated this method using photoactive yellow protein (PYP) from Halorhodospira halophila, a prototype of the Per-Arnt-Sim (PAS) domain superfamily that contains only two native histidine residues. We tested both the efficiency of isotope editing of histidine and the degree of isotope scrambling after protein overproduction in E. coli grown in defined growth medium containing the 20 amino acids. Using varied amounts of isotope-edited histidine in the range of 0, 8, 16, 32, 64, and 128 mg/L of cell culture, we found that 32 mg of histidine per liter of cell culture is sufficient for over 98% of isotope labeling using double ionization mass spectroscopy measurements. Analysis of isotope envelopes of mass spectra of intact PYP are consistent with the absence of isotope scrambling, as are nuclear magnetic resonance spectra that show only resonances from the labeled histidine residues. In addition, we use Fourier transform infrared spectroscopy to demonstrate that these protein samples allow the detection of vibrational modes shifted upon histidine-specific isotope labeling. This method is widely applicable to histidine-specific isotope labeling of other proteins upon overexpression in E. coli.