The use of unnatural amino acids to study and engineer protein function

Neumann‐Staubitz, Petra; Neumann, Heinz

doi:10.1016/j.sbi.2016.06.006

Cited by 97 publications

(65 citation statements)

References 77 publications

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“…Consequently,c hemical probesh ave great potentialt oo vercome challenges in the field, including those that have not yet been applied to O-GlcNAc-cycling enzymes( such as unnatural amino acids). [72] By improving the specificity,s ensitivity,a nd efficiency of the labeling and detection methods,c hemical probesa re promising tools for cellular or in vivo studies.…”

Section: Microarrays To Elucidate the Substrate Preferences Of Ogt Anmentioning

confidence: 99%

“…Overall, small‐molecule probes are versatile as they can be modified with different functional groups to tune their reactivity or to apply them in entirely new strategies. Consequently, chemical probes have great potential to overcome challenges in the field, including those that have not yet been applied to O ‐GlcNAc‐cycling enzymes (such as unnatural amino acids) . By improving the specificity, sensitivity, and efficiency of the labeling and detection methods, chemical probes are promising tools for cellular or in vivo studies.…”

Section: Introductionmentioning

confidence: 99%

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Chemical and Biochemical Strategies To Explore the Substrate Recognition of O‐GlcNAc‐Cycling Enzymes

Worth

et al. 2018

ChemBioChem

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The O-linked N-acetylglucosamine (O-GlcNAc) modification is an essential component in cell regulation. A single pair of human enzymes conducts this modification dynamically on a broad variety of proteins: O-GlcNAc transferase (OGT) adds the GlcNAc residue and O-GlcNAcase (OGA) hydrolyzes it. This modification is dysregulated in many diseases, but its exact effect on particular substrates remains unclear. In addition, no apparent sequence motif has been found in the modified proteins, and the factors controlling the substrate specificity of OGT and OGA are largely unknown. In this minireview, we will discuss recent developments in chemical and biochemical methods toward addressing the challenge of OGT and OGA substrate recognition. We hope that the new concepts and knowledge from these studies will promote research in this area to advance understanding of O-GlcNAc regulation in health and disease.

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Section: Microarrays To Elucidate the Substrate Preferences Of Ogt Anmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Chemical and Biochemical Strategies To Explore the Substrate Recognition of O‐GlcNAc‐Cycling Enzymes

Worth

et al. 2018

ChemBioChem

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“…Genetic code expansion (GCE) incorporates artificial amino acids into polypeptide chains to create synthetic proteins with novel functions; it has many applications, ranging from discovery science to molecular medicine [52][53][54]. In GCE, an orthogonal tRNA/cognate synthetase pair is used to suppress a rare stop codon introduced at a specific site in a gene of interest.…”

Section: Multibactag: Extending the Scope Of Genetic Code Expansionmentioning

confidence: 99%

MultiBac: from protein complex structures to synthetic viral nanosystems

et al. 2017

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The MultiBac baculovirus/insect cell expression vector system was conceived as a user-friendly, modular tool-kit for producing multiprotein complexes for structural biology applications. MultiBac has allowed the structure and function of many molecular machines to be elucidated, including previously inaccessible high-value drug targets. More recently, MultiBac developments have shifted to customized baculoviral genomes that are tailored for a range of applications, including synthesizing artificial proteins by genetic code expansion. We review some of these developments, including the ongoing rewiring of the MultiBac system for mammalian applications, notably CRISPR/Cas9-mediated gene editing.

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“…This methodology, commonly referred to as amber suppression, relied on the development of an orthogonal tRNA/aaRS pair that could be expressed in E. coli and was used to incorporate O -methyl-L-tyrosine into dihydrofolate reductase with 99% fidelity. In the 15 years since this landmark study, the unnatural building block palette for genetic incorporation has grown more than 100 amino acids strong (Lang & Chin, 2014; Liu & Schultz, 2010; Neumann-Staubitz & Neumann, 2016). This includes amino acids carrying natural modifications (e.g.…”

Section: Molecular Engineering Toolbox For Complex Biological Samplesmentioning

confidence: 99%

A molecular engineering toolbox for the structural biologist

Debelouchina

Muir

2017

Quart. Rev. Biophys.

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Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease.

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The use of unnatural amino acids to study and engineer protein function

Cited by 97 publications

References 77 publications

Chemical and Biochemical Strategies To Explore the Substrate Recognition of O‐GlcNAc‐Cycling Enzymes

Chemical and Biochemical Strategies To Explore the Substrate Recognition of O‐GlcNAc‐Cycling Enzymes

MultiBac: from protein complex structures to synthetic viral nanosystems

A molecular engineering toolbox for the structural biologist

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