1 research outputs found
Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy
Vibrational
spectra contain unique information on protein structure
and dynamics. However, this information is often obscured by spectral
congestion, and site-selective information is not available. In principle,
sites of interest can be spectrally identified by isotope shifts,
but site-specific isotope labeling of proteins is today possible only
for favorable amino acids or with prohibitively low yields. Here we
present an efficient cell-free expression system for the site-specific
incorporation of any isotope-labeled amino acid into proteins. We
synthesized 1.6 mg of green fluorescent protein with an isotope-labeled
tyrosine from 100 mL of cell-free reaction extract. We unambiguously
identified spectral features of the tyrosine in the fingerprint region
of the time-resolved infrared absorption spectra. Kinetic analysis
confirmed the existence of an intermediate state between photoexcitation
and proton transfer that lives for 3 ps. Our method lifts vibrational
spectroscopy of proteins to a higher level of structural specificity