分类: 生物学 >> 生物物理学 提交时间: 2016-05-18
摘要: Photo-induced electron transfer (PET) is ubiquitous for photosynthesis and fluorescent sensor design. However, genetically coded PET sensors are underdeveloped, due to the lack of methods to site-specifically install PET probes on proteins. Here we describe a family of acid and Mn(III) turn-on fluorescent protein (FP) sensors, named iLovU, based on PET and the genetic incorporation of superior PET quenchers in the fluorescent flavoprotein iLov. Using the iLovU PET sensors, we monitored the cytoplasmic acidification process, and achieved Mn(III) fluorescence sensing for the first time. The iLovU sensors should be applicable for studying pH changes in living cells, monitoring biogentic Mn(III) in the environment, and screening for efficient manganese peroxidase, which is highly desirable for lignin degradation and biomass conversion. Our work establishes a platform for many more protein PET sensors, facilitates the de novo design of metalloenzymes harboring redox active residues, and expands our ability to probe protein conformational dynamics.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-11
摘要: Lysine formylation is a newly discovered post-translational modification (PTM) in histones and other nuclear proteins; it has a well-recognized but poorly defined role in chromatin conformation modulation and gene expression. To date, there is no general method to site-specifically incorporate N-epsilon-formyllysine at a defined site of a protein. Here we report the highly efficient genetic incorporation of the unnatural amino acid N-epsilon-formyllysine into proteins produced in Escherichia coli and mammalian cells, by using an orthogonal N-epsilon-formyllysine tRNAsynthetase/tRNA(CUA) pair. This technique can be applied to study the role of lysine formylation in epigenetic regulation.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both invitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site- and sequence-specific covalent labeling of RNAs in mammalian cells by using tRNA(Ile2)-agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide-bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site-specific transfer of azide/alkyne groups to an RNA molecule of interest invitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA.