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Reversible labeling of native and fusion-protein motifs.

ABSTRACT: The reversible covalent attachment of chemical probes to proteins has long been sought as a means to visualize and manipulate proteins. Here we demonstrate the full reversibility of post-translational custom pantetheine modification of Escherichia coli acyl carrier protein for visualization and functional studies. We use this iterative enzymatic methodology in vitro to reversibly label acyl carrier protein variants and apply these tools to NMR structural studies of protein-substrate interactions.

SUBMITTER: Kosa NM

PROVIDER: S-EPMC4128096 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Reversible labeling of native and fusion-protein motifs.

Kosa Nicolas M NM Haushalter Robert W RW Smith Andrew R AR Burkart Michael D MD

Nature methods 20120916 10

The reversible covalent attachment of chemical probes to proteins has long been sought as a means to visualize and manipulate proteins. Here we demonstrate the full reversibility of post-translational custom pantetheine modification of Escherichia coli acyl carrier protein for visualization and functional studies. We use this iterative enzymatic methodology in vitro to reversibly label acyl carrier protein variants and apply these tools to NMR structural studies of protein-substrate interactions ...[more]

PMID: 22983458

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Project description:Site-specific chemical modification of proteins is important for many applications in biology and biotechnology. Recently, our laboratory and others have exploited the high specificity of the enzyme protein farnesyltransferase (PFTase) to site-specifically modify proteins through the use of alternative substrates that incorporate bioorthogonal functionality including azides and alkynes. In this study, we evaluate two aldehyde-containing molecules as substrates for PFTase and as reactants in both oxime and hydrazone formation. Using green fluorescent protein (GFP) as a model system, we demonstrate that the purified protein can be enzymatically modified with either analogue to yield aldehyde-functionalized proteins. Oxime or hydrazone formation was then employed to immobilize, fluorescently label, or PEGylate the resulting aldehyde-containing proteins. Immobilization via hydrazone formation was also shown to be reversible via transoximization with a fluorescent alkoxyamine. After characterizing this labeling strategy using pure protein, the specificity of the enzymatic process was used to selectively label GFP present in crude E. coli extract followed by capture of the aldehyde-modified protein using hydrazide-agarose. Subsequent incubation of the immobilized protein using a fluorescently labeled or PEGylated alkoxyamine resulted in the release of pure GFP containing the desired site-specific covalent modifications. This procedure was also employed to produce PEGylated glucose-dependent insulinotropic polypeptide (GIP), a protein with potential therapeutic activity for diabetes. Given the specificity of the PFTase-catalyzed reaction coupled with the ability to introduce a CAAX-box recognition sequence onto almost any protein, this method shows great potential as a general approach for selective immobilization and labeling of recombinant proteins present in crude cellular extract without prior purification. Beyond generating site-specifically modified proteins, this approach for polypeptide modification could be particularly useful for large-scale production of protein conjugates for therapeutic or industrial applications.

Dataset Information

Reversible labeling of native and fusion-protein motifs.

Publications

Reversible labeling of native and fusion-protein motifs.

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