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

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.

A proposal for a dipole-generated BLUF domain mechanism

“…The presence of an unusually strong hydrogen bond donated from Tyr-21 in the signaling state of AppA was interpreted as incompatible with hydrogen bonding to the glutamine side chain amidic nitrogen (Iwata et al, 2011 ), while a previous study on TePixD concluded that both dark and light state properties of tyrosine are compatible with H-bonding to oxygen but could not fully exclude alternative orientations (Takahashi et al, 2007 ). A study on SyPixD with a different tyrosine isotope labeling pattern gave similar results and clearly showed that the structural changes are confined to the phenolic hydroxyl group without any changes to the aromatic nature of the phenyl ring (Mehlhorn et al, 2013 ).…”

Light‐Induced Conformational Changes in the Plant Cryptochrome Photolyase Homology Region Resolved by Selective Isotope Labeling and Infrared Spectroscopy

“…Escherichia coli CpXribF cells were transformed by electroporation with the plasmid pET11a‐CPH1‐PHR encoding amino acids 1‐504 of the plant cryptochrome from Chlamydomonas reinhardtii and an N‐terminal Strep tag II . Cells were grown in M9 minimal medium supplemented with 200 mg/L ampicillin and 50 μ m riboflavin following published procedures .…”

“…In contrast, reconstitution of photolyases or cryptochromes with FAD has been unsuccessful to date, calling for a different strategy. Recently, such an approach has been developed for heterologous expression in Escherichia coli , which includes a shutdown of the cellular riboflavin production, an enhancement of riboflavin intake by a transporter and the increase in FAD production in vivo by a bifunctional flavokinase/adenylase . Thereby, flavin at natural isotope abundance is incorporated during expression (Fig.…”

“…Thereby, flavin at natural isotope abundance is incorporated during expression (Fig. ), which has been demonstrated to lead to valuable insight by infrared spectroscopy on the mechanism of BLUF (blue light sensor using flavin) domains and CPD photolyases .…”

Synthesis and application of isotopically labeled flavin nucleotides

“…Here we present the synthesis of several flavin nucleotides isotopically labeled at the isoalloxazine core or the adenyl tail, and their utility in analyzing an intermediate of an enzymatic reaction. Isotopically labeled flavins and flavin analogues have been used in the past to probe the structural dynamics of light receptors, elucidate flavin transport and metabolism in healthy and diseased cells and gain insight into the mechanisms of flavoproteins, among other applications. In particular, reconstitution of cytochrome P450 reductase, the redox partner of P450 enzymes, with FMN analogues shed light on the mechanism of electron transfer in this protein .…”