Side-chain specific isotopic labeling of proteins for infrared structural biology: the case of ring-D4-tyrosine isotope labeling of photoactive yellow protein.
An important bottleneck in the use of infrared spectroscopy as a powerful tool for obtaining detailed information on protein structure is the assignment of vibrational modes to specific amino acid residues. Side-chain specific isotopic labeling is a general approach towards obtaining such assignments. We report a method for high yield isotope editing of the bacterial blue light sensor photoactive yellow protein (PYP) containing ring-D(4)-Tyr. PYP was heterologously overproduced in Escherichia coli in minimal media containing ring-D(4)-Tyr in the presence of glyphosate, which inhibits endogenous biosynthesis of aromatic amino acids (Phe, Trp, and Tyr). Mass spectrometry of the intact protein and of tryptic peptides unambiguously demonstrated highly specific labeling of all five Tyr residues in PYP with 98% incorporation and undetectable isotopic scrambling. FTIR spectroscopy of the protein reveals a characteristic Tyr ring vibrational mode at 1515 cm(-1) that is shifted to 1436 cm(-1), consistent with that from ab initio calculations. PYP is a model system for protein structural dynamics and for receptor activation in biological signaling. The results described here open the way to the analysis of PYP using isotope-edited FTIR spectroscopy with side-chain specific labeling.
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA.
Rachana
R
Rathod
Zhouyang
Z
Kang
Steven D
SD
Hartson
Masato
M
Kumauchi
Aihua
A
Xie
Wouter D
WD
Hoff
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D020607:Halorhodospira halophila / Q000737:chemistry* / Q000235:genetics*; D007553:Isotope Labeling; D013058:Mass Spectrometry; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics*; D017550:Spectroscopy, Fourier Transform Infrared; D014443:Tyrosine / Q000737:chemistry*; D015854:Up-Regulation
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria; D014443:Tyrosine