Resin supported acyl carrier protein labeling strategies

Rothmann, Michael; Kosa, Nicolas M.; Burkart, Michael D.

doi:10.1039/c3ra47847e

Cited by 4 publications

(4 citation statements)

References 26 publications

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“…Furthermore, NMR-based experiments on these short peptide tags identified six key amino acids critical for interaction with the enzyme. 280 That allowed the design of an 8-residue peptide, A4 (DSLDMLEW) containing 5 of the 6 key residues that displayed comparable efficiency to that of A1 in AcpS-mediated protein labelling.…”

Section: Enzymatic Protein Labelling Strategiesmentioning

confidence: 99%

Recent progress in enzymatic protein labelling techniques and their applications

et al. 2018

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Protein-based conjugates are valuable constructs for a variety of applications. Conjugation of proteins to fluorophores is commonly used to study their cellular localization and the protein-protein interactions. Modification of therapeutic proteins with either polymers or cytotoxic moieties greatly enhances their pharmacokinetics and potency. To label a protein of interest, conventional direct chemical reaction with the side-chains of native amino acids often yields heterogeneously modified products. This renders their characterization complicated, requires difficult separation steps and may impact protein function. Although modification can also be achieved via the insertion of unnatural amino acids bearing bioorthogonal functional groups, these methods can have lower protein expression yields, limiting large scale production. As a site-specific modification method, enzymatic protein labelling is highly efficient and robust under mild reaction conditions. Significant progress has been made over the last five years in modifying proteins using enzymatic methods for numerous applications, including the creation of clinically relevant conjugates with polymers, cytotoxins or imaging agents, fluorescent or affinity probes to study complex protein interaction networks, and protein-linked materials for biosensing. This review summarizes developments in enzymatic protein labelling over the last five years for a panel of ten enzymes, including sortase A, subtiligase, microbial transglutaminase, farnesyltransferase, N-myristoyltransferase, phosphopantetheinyl transferases, tubulin tyrosin ligase, lipoic acid ligase, biotin ligase and formylglycine generating enzyme.

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Section: Enzymatic Protein Labelling Strategiesmentioning

confidence: 99%

Recent progress in enzymatic protein labelling techniques and their applications

et al. 2018

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“…This poses an interesting challenge for future studies in which we can envision optimizing chemoenzymatic labeling by engineering the protein array surfaces, the peptide substrates (leveraging machine learning and optimization tools 45 ), and the Sfp enzyme for complementary electrostatic interactions or improved reaction sterics. Another exciting possibility is the use of secondary enzymes (like acyl carrier protein hydrolases), 43,66 which have been shown to selectively cleave ybbR peptide tags, opening the path for the reversible tailoring of protein array surfaces. Enzyme-mediated post-translational modification of proteins is ubiquitous in nature and greatly increases the complexity of the proteome.…”

Section: ■ Conclusionmentioning

confidence: 99%

Enzyme-Directed Functionalization of Designed, Two-Dimensional Protein Lattices

et al. 2020

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The design and construction of crystalline protein arrays to selectively assemble ordered nanoscale materials have potential applications in sensing, catalysis, and medicine. Whereas numerous designs have been implemented for the bottom-up construction of protein assemblies, the generation of artificial functional materials has been relatively unexplored. Enzyme-directed post-translational modifications are responsible for the functional diversity of the proteome and, thus, could be harnessed to selectively modify artificial protein assemblies. In this study, we describe the use of phosphopantetheinyl transferases (PPTases), a class of enzymes that covalently modify proteins using coenzyme A (CoA), to site-selectively tailor the surface of designed, two-dimensional (2D) protein crystals. We demonstrate that a short peptide (ybbR) or a molecular tag (CoA) can be covalently tethered to 2D arrays to enable enzymatic functionalization using Sfp PPTase. The site-specific modification of two different protein array platforms is facilitated by PPTases to afford both small molecule- and protein-functionalized surfaces with no loss of crystalline order. This work highlights the potential for chemoenzymatic modification of large protein surfaces toward the generation of sophisticated protein platforms reminiscent of the complex landscape of cell surfaces.

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“…[48] Both resin-attached and free protein techniques have been developed, allowing for the preparation of large quantities of apo -ACP and the recycling of high-value, isotopically enriched ACPs for NMR use. [49,50] Reversible tagging has also allowed for quantitative “apo-fication” of holo - and crypto-ACPs, yielding homogenous apo -ACP for further modification (Figure 2b). …”

Section: The Acyl Carrier Protein – An Extended Toolkitmentioning

confidence: 99%

Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

2015

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Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in Nature, preserving the same handful of chemical reactions over all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock or antimicrobial purposes has been met with limited success in part due to a lack of detailed molecular information behind the ACP-partner protein interactions inherent to the pathway. This review will focus on recently developed tools for the modification of ACP and analysis of protein-protein interactions, such as mechanism-based crosslinking, and the studies exploiting them. Discussion specific to each enzymatic domain focuses first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. While significant unknowns remain, new understandings into the intricacies of FAS point to future advances in manipulating this complex molecular factory.

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Resin supported acyl carrier protein labeling strategies

Cited by 4 publications

References 26 publications

Recent progress in enzymatic protein labelling techniques and their applications

Recent progress in enzymatic protein labelling techniques and their applications

Enzyme-Directed Functionalization of Designed, Two-Dimensional Protein Lattices

Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

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