Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy

Abstract

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.

Watching Proteins Wiggle: Mapping Structures with Two-Dimensional Infrared Spectroscopy

“…30 Recently, Peuker et al showed that non-natural isotope labels can be included in large proteins using cell-free expression. 89 These latter two methods are extremely exciting, as they have opened the door to isotope labeling proteins regardless of size.…”

Site‐Specific Isotopic Labeling (SSIL): Access to High‐Resolution Structural and Dynamic Information in Low‐Complexity Proteins

“…with amino acids, nucleotides, salts and an energy regeneration system ( Figure 2). [20,21,28,30,31] It is important to mention that, once used, the tRNA CUA cannotb er eloaded inside the CF reaction, making this process ao ne-off reaction.…”

“…[30] More recently,t he green fluorescent protein (GFP) was produced with as ingle [ 13 C, 18 O]tyrosine. [31] The sample enabledt he recording of timeresolved infrared absorption spectra in as ite-specific manner and yieldedn ovel information on the photodynamicso fG FP. Despite these successful examples, when aiming to use isotopically labeled canonical amino acids, the synthesis of large quantities of the aminoacylated dinucleotides presentsa ni mportant bottleneck.…”

“…The loaded tRNA CUA is then added to a cell‐free (CF) system where the target protein is produced. CF is an in vitro protein synthesis method composed of a lysate containing the transcription‐translation machinery, normally obtained from E. coli cultures, which is supplemented with amino acids, nucleotides, salts and an energy regeneration system (Figure ) . It is important to mention that, once used, the tRNA CUA cannot be reloaded inside the CF reaction, making this process a one‐off reaction.…”

“…In a pioneering example, the semisynthetic approach was used to load a tRNA CUA with a [ 13 C]alanine, yielding a highly simplified 13 C‐filtered 1 H spectrum of T4 lysozyme in native and denaturing conditions . More recently, the green fluorescent protein (GFP) was produced with a single [ 13 C, 18 O]tyrosine . The sample enabled the recording of time‐resolved infrared absorption spectra in a site‐specific manner and yielded novel information on the photodynamics of GFP.…”