A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors

Jennifer Mehlhorn,Sebastian Beck,Tilo Mathes,John T. M. Kennis,Jose M. Sanchez-Ruiz,Peter Hegemann,Helena Steinocher

doi:10.1371/journal.pone.0079006

Jennifer Mehlhorn, Sebastian Beck

Open Access

https://doi.org/10.1371/journal.pone.0079006

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Abstract

Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.

Highlights

Since the advent of cloning in the early 1970s heterologous overexpression of proteins has become the method of choice for studying protein function on a molecular level
NMR is able to visualize the dynamic fluctuations of proteins down to the picosecond time domain [1,2], light- or chemical induced reactions that occur on a sub millisecond timescale likesensory processes or single enzymatic turnovers may not be followed in real time, while vibrational spectroscopy can be performed in real time with femtosecond resolution instead [3,4]
Compared to site-directed mutagenesis isotope labeling is a non-invasive protein modification and allows the clear identification and assignment of vibrational signatures by comparison of spectroscopic data from labeled and unlabeled variants. In this regard the labeling efficiency is especially important for vibrational spectroscopy, since contributions of labeled and unlabeled proteins are recorded at the same time

Summary

Introduction

Since the advent of cloning in the early 1970s heterologous overexpression of proteins has become the method of choice for studying protein function on a molecular level. Since no functional mutation of the glutamine side chain is possible and the vibrational signatures of flavin carbonyl groups and glutamine amide side chain are expected to overlap, a direct proof of either model may only be obtained by vibrational spectroscopy on selectively flavin or amino acid side chain isotope labeled BLUF domains. For both IR and NMR spectroscopy global labeling approaches have been long established [27]. We illustrate the potential of this approach by presenting FTIR lightminus-dark difference spectra of the Synechocystis BLUF photoreceptor Slr1694 (SyPixD) with selective flavin and protein labeling patterns

Experimental Procedures

Results and Discussion

Conclusions and Perspective