ABSTRACT
Posttranslational modification (PTM) is a key mechanism for regulating diverse protein functions, and thus critically affects many essential biological processes. Critical for systematic study of the effects of PTMs is the ability to obtain recombinant proteins with defined and homogenous modifications. To this end, various synthetic and chemical biology approaches, including genetic code expansion and protein chemical modification methods, have been developed. These methods have proven effective for generating site-specific authentic modifications or structural mimics, and have demonstrated their value for in vitro and in vivo functional studies of diverse PTMs. This review will discuss recent advances in chemical biology strategies and their application to various PTM studies.
Subject(s)
Chemistry Techniques, Synthetic/methods , Genetic Code , Protein Processing, Post-Translational , Proteome/metabolism , Acetylation , Codon, Terminator/chemistry , Codon, Terminator/metabolism , Glycosylation , Humans , Lysine/analogs & derivatives , Lysine/metabolism , Methylation , Nitrates/metabolism , Phosphorylation , Proteome/genetics , Selenocysteine/metabolism , Sulfates/metabolism , UbiquitinationABSTRACT
The lysine acetylation of proteins plays a key role in regulating protein functions, thereby controlling a wide range of cellular processes. Despite the prevalence and significance of lysine acetylation in eukaryotes, however, its systematic study has been challenged by the technical limitations of conventional approaches for selective lysine acetylation in vivo. Here, we report the in vivo study of lysine acetylation via the genetic incorporation of Nε-acetyllysine in yeast. We demonstrate that a newly discovered acetylation-sumoylation switch precisely controls the localization and cellular function of the yeast septin protein, Cdc11, during the cell cycle. This approach should facilitate the comprehensive in vivo study of lysine acetylation across a wide range of proteins in eukaryotic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.
Subject(s)
Acetylation , Lysine/metabolism , Protein Engineering/methods , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sumoylation , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cytokinesis/genetics , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Genetic Complementation Test , Lysine/genetics , Protein Processing, Post-Translational , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Synthetic Biology/methodsABSTRACT
Analysis of protein dynamics using single-molecule fluorescence resonance energy transfer (smFRET) is widely used to understand the structure and function of proteins. Nonetheless, site-specific labeling of proteins with a pair of donor and acceptor dyes still remains a challenge. Here we present a general and facile method for site-specific dual labeling of proteins by incorporating two different, readily available, unnatural amino acids (p-acetylphenylalanine and alkynyllysine) for smFRET. We used newly evolved alkynyllysine-specific aminoacyl-tRNA synthetase/tRNA(UCA) and p-acetylphenylalanyl-tRNA synthetase/tRNA(CUA). The utility of our approach was demonstrated by analyzing the conformational change of dual-labeled calmodulin using smFRET measurements. The present labeling approach is devoid of major limitations in conventional cysteine-based labeling. Therefore, our method will significantly increase the repertoire of proteins available for FRET study and expand our ability to explore more complicated molecular dynamics.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Proteins/metabolism , RNA, Transfer/metabolism , Base Sequence , Binding Sites/physiology , Molecular Sequence Data , Protein Structure, Secondary , Proteins/chemistry , Proteins/genetics , RNA, Transfer/chemistry , RNA, Transfer/geneticsABSTRACT
Despite the importance of glucose and amino acids for energy metabolism, interactions between the two nutrients are not well understood. We provide evidence for a role of leucyl-tRNA synthetase 1 (LARS1) in glucose-dependent control of leucine usage. Upon glucose starvation, LARS1 was phosphorylated by Unc-51 like autophagy activating kinase 1 (ULK1) at the residues crucial for leucine binding. The phosphorylated LARS1 showed decreased leucine binding, which may inhibit protein synthesis and help save energy. Leucine that is not used for anabolic processes may be available for catabolic pathway energy generation. The LARS1-mediated changes in leucine utilization might help support cell survival under glucose deprivation. Thus, depending on glucose availability, LARS1 may help regulate whether leucine is used for protein synthesis or energy production.
Subject(s)
Energy Metabolism , Glucose/metabolism , Leucine-tRNA Ligase/metabolism , Leucine/metabolism , Animals , Autophagy-Related Protein-1 Homolog/genetics , Autophagy-Related Protein-1 Homolog/metabolism , Fibroblasts , HEK293 Cells , HeLa Cells , Humans , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Monomeric GTP-Binding Proteins/metabolism , Phosphorylation , Signal TransductionABSTRACT
We present a new platform for multiplexed protein kinase activity assay using TiO2 decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening. On the basis of the strong affinity of TiO2 for the phosphate group and the fluorescence quenching capability of GO, phosphorylation of substrates by protein kinases was quantitatively measured in a short time.