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Michael D Burkart

Publications and source records attributed to Michael D Burkart.

At least 19 recordsLinked to original sources
PubMed2006

Site-specific protein modification: advances and applications.

Although chemical methods to modify proteins in a sequence-specific manner have yet to be developed, site-specific post-translational modification of proteins has recently emerged as a major focus in biological chemistry. Post-translational modification with functionalized substrate analogues opens up several unique avenues to induce selective reactivity into proteins in a sequence-specific manner, and can be applied to protein identification and manipulation in both in vitro and in vivo contexts. Further in vivo applications of this method will enable the imaging of cellular processes, avoiding nonspecific labeling and probe scattering, major complications observed in nonenzymatic methods. Additionally, new tools for in vitro protein modification have been developed that offer more versatile ways to study protein structure and function.

Mutagenesis, Site-Directed↗
PubMed2006

Mechanism-based protein cross-linking probes to investigate carrier protein-mediated biosynthesis.

Fatty acid, polyketide, and nonribosomal peptide biosynthetic enzymes perform structural modifications upon small molecules that remain tethered to a carrier protein. This manuscript details the design and analysis of cross-linking substrates that are selective for acyl carrier proteins and their cognate condensing enzymes. These inactivators are engineered through a covalent linkage to fatty acid acyl carrier protein via post-translational modification to contain a reactive probe that traps the active site cysteine residue of ketosynthase domains. These proteomic tools are applied to Escherichia coli fatty acid synthase enzymes, where KASI and KASII selectively cross-link ACP-bound epoxide and chloroacrylate moieties. These mechanism-based, protein-protein fusion reagents also demonstrated cross-linking of KASI to type II polyketide ACPs, while nonribosomal peptide carrier proteins showed no reactivity. Similar investigations into protein-protein interactions, proximity effects, and substrate specificities will be required to complete the mechanistic understanding of these pathways.

Carrier Proteins↗
PubMed2006

A systems perspective to digital structures in molecular analysis.

Within the last decade, the advance of miniaturization has opened the window to new systems that permit digital and molecular science to intersect, suggesting a new role for organic chemistry. Currently, fusion of molecules and electronic digits, as well as molecular-based digital structures, have expanded the conventional interpretation of the digit. This emergence has already generated new technological platforms with unique applications for molecular analysis and computation. We provide a brief overview of the conventional understanding of digital devices, examine the concept of molecular-based digits, and suggest new architectures by examining studies conducted on the compact discs. This analysis presents a perspective for the unique interaction of molecules and digits and the development of digital-based platforms for molecular analysis.

Journal Article↗
PubMed2006

Synthesis and evaluation of bioorthogonal pantetheine analogues for in vivo protein modification.

In vivo carrier protein tagging has recently become an attractive target for the site-specific modification of fusion systems and new approaches to natural product proteomics. A detailed study of pantetheine analogues was performed in order to identify suitable partners for covalent protein labeling inside living cells. A rapid synthesis of pantothenamide analogues was developed and used to produce a panel which was evaluated for in vitro and in vivo protein labeling. Kinetic comparisons allowed the construction of a structure-activity relationship to pinpoint the linker, dye, and bioorthogonal reporter of choice for carrier protein labeling. Finally bioorthogonal pantetheine analogues were shown to target carrier proteins with high specificity in vivo and undergo chemoselective ligation to reporters in crude cell lysate. The methods demonstrated here allow carrier proteins to be visualized and isolated for the first time without the need for antibody techniques and set the stage for the future use of carrier protein fusions in chemical biology.

Bacterial Proteins↗
PubMed2006

Geometry in digital molecular arrays.

The development of digital molecular devices arises through the appropriate geometric positioning of a molecular assay. A detailed evaluation of the digital media reveals the critical aspects of geometric positioning in terms of developing an analytically-robust system for molecular analysis. This study reveals an explicit digital compact disc based assay for molecular affinity events.

Molecular Structure↗
PubMed2005

One-pot chemo-enzymatic synthesis of reporter-modified proteins.

To meet recent advancements in the covalent reporter labeling of proteins, we propose a flexible synthesis for reporter analogs. Here we demonstrate a one-pot chemo-enzymatic synthesis of reporter-labeled proteins that allows the covalent tethering of any amine-terminal fluorescent or affinity label to a carrier protein or fusion construct. This two-reaction sequence consists of activated panthothenate coupling, biosynthetic conversion to the coenzyme A (CoA) analog, and enzymatic carrier protein modification via phosphopantetheinyltransferase (PPTase). We also probe substrate specificity for CoAA, the first enzyme in the pathway. With this approach CoA analogs may be rapidly prepared, thus permitting the regiospecific attachment of reporter moieties from a variety of molecular species.

Affinity Labels↗
PubMed2005

In vivo reporter labeling of proteins via metabolic delivery of coenzyme A analogues.

We demonstrate site-specific reporter labeling of proteins within live cells. By accessing the coenzyme A (CoA) metabolic pathway with a cell-permeable pantetheine analogue, we deliver CoA-bound reporter molecules for post-translational protein modification in vivo. These methods may be applied to natural product pathway manipulation as well as applications in conventional molecular and cellular biology.

Carrier Proteins↗
PubMed2005

A three-component photoreversible tag for thiols.

[structure: see text] A one-pot coupling of a 1,3-diketone, an aldehyde, and an alkanethiol has been developed to produce a protected sulfide. Through use of an o-nitrophenylbenzaldehyde, this method provides a one-step route to a photochemically reversible thiol-protecting group. The kinetics of photolysis were established using (1)H NMR analysis, which allows for the rate to be based on the entire reaction scheme.

Journal Article↗
PubMed2004

Selectfluor: mechanistic insight and applications.

The replacement of hydrogen atoms with fluorine substituents in organic substrates is of great interest in synthetic chemistry because of the strong electronegativity of fluorine and relatively small steric footprint of fluorine atoms. Many sources of nucleophilic fluorine are available for the derivatization of organic molecules under acidic, basic, and neutral conditions. However, electrophilic fluorination has historically required molecular fluorine, whose notorious toxicity and explosive tendencies limit its application in research. The necessity for an electrophilic fluorination reagent that is safe, stable, highly reactive, and amenable to industrial production as an alternative to very hazardous molecular fluorine was the inspiration for the discovery of selectfluor. This reagent is not only one of the most reactive electrophilic fluorinating reagents available, but it is also safe, nontoxic, and easy to handle. In this Review we document the many applications of selectfluor and discuss possible mechanistic pathways for its reaction.

Diazonium Compounds↗
PubMed2004

Modular synthesis of pantetheine and phosphopantetheine.

[structure: see text] D-Pantetheine and D-phosphopantetheine, precursors to coenzyme A, have been synthesized though a linear sequence from three modules (M1-M3) in 9 and 10 steps, respectively. These routes provide access to analogues of coenzyme A containing modified cystamines, beta-alanines, and pantoic acid residues. All three modules were joined using conventional methods of peptide synthesis. The chiral component, M3, was derived from D-pantolactone.

Molecular Structure↗
PubMed2004

The position of a key tyrosine in dTDP-4-Keto-6-deoxy-D-glucose-5-epimerase (EvaD) alters the substrate profile for this RmlC-like enzyme.

Vancomycin, the last line of defense antibiotic, depends upon the attachment of the carbohydrate vancosamine to an aglycone skeleton for antibacterial activity. Vancomycin is a naturally occurring secondary metabolite that can be produced by bacterial fermentation. To combat emerging resistance, it has been proposed to genetically engineer bacteria to produce analogues of vancomycin. This requires a detailed understanding of the biochemical steps in the synthesis of vancomycin. Here we report the 1.4 A structure and biochemical characterization of EvaD, an RmlC-like protein that is required for the C-5' epimerization during synthesis of dTDP-epivancosamine. EvaD, although clearly belonging to the RmlC class of enzymes, displays very low activity in the archetypal RmlC reaction (double epimerization of dTDP-6-deoxy-4-keto-D-glucose at C-3' and C-5'). The high resolution structure of EvaD compared with the structures of authentic RmlC enzymes indicates that a subtle change in the enzyme active site repositions a key catalytic Tyr residue. A mutant designed to re-establish the normal position of the Tyr increases the RmlC-like activity of EvaD.

Amino Acid Sequence↗
PubMed2004

Manipulation of carrier proteins in antibiotic biosynthesis.

Engineering biosynthetic pathways into suitable host organisms has become an attractive venue for the design, evaluation, and production of small molecule therapeutics. Polyketide (PK) and nonribosomal peptide (NRP) synthases have been of particular interest due to their modular structure, yet routine cloning and expression of these enzymes remains challenging. Here we describe a method to covalently label carrier proteins from PK and NRP synthases using the enzymatic transfer of a modified coenzyme A analog by a 4'-phosphopantetheinyltransferase. Using this method, carrier proteins can be loaded with single fluorescent or affinity reporters, providing novel entry for protein visualization, Western blot identification, and affinity purification. Application of these methods provides an ideal tool to track and quantify metabolically engineered pathways. Such techniques are valuable to measure protein expression, solubility, activity, and native posttranslational modification events in heterologous systems.

Anti-Bacterial Agents↗
PubMed2003

Molecular screening on a compact disc.

A method is described to screen the recognition between small molecule ligands and biomolecules using a conventional compact disc (CD) player. A procedure was developed to attach ligands to the reading face of a CD by activating the terminus of polycarbonate, a common polymer composite within the reading face of a CD. Terminal residues of the polycarbonate surface 1 were phosphorylated by reaction with ethyl-(N,N)-diisopropylamine-buffered dichloro-(N,N)-diisopropylaminophosphate to yield surface-bound chlorophosphate 2. Ligands containing a primary alcohol were condensed with 2 providing a polycarbonate capped with phosphodiester linked ligands 3-6. Displays were generated on the surface of a CD by printing tracks of ligands 3-6 on the disc with an inkjet printer. Using this method, discs were created with entire assemblies of ligand molecules distributed into separate blocks. A molecular array was developed by assembling collections of these blocks to correlate with the CDROM-XA formatted data stored within the digital layer of the disc. Regions of the disc containing a given ligand or set of ligands was marked by its spatial position using the tracking and header information. Recognition between surface expressed ligands and biomolecules was screened by an error determination routine.

Journal Article↗