- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Observation of Site-Resolved Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater
摘要： Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, however, is sparse because site-selective injection of vibrational energy into a protein, i.e. localized heating, is required for their investigation. Here, we solve this problem by the site-specific incorporation of the non-canonical amino acid β-(1-azulenyl)-L-alanine (AzAla) via genetic code expansion. Being an exception to Kasha′s rule, AzAla undergoes ultrafast internal conversion and heating after S1 excitation while upon S2 excitation it serves as a fluorescent label. We endowed PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. Using ultrafast IR spectroscopy, we could indeed observe VET from the incorporation sites in the protein to a bound peptide ligand on a picosecond timescale. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role for function in a wide range of proteins.
关键词： protein modification,energy transfer,non-canonical amino acid,time-resolved spectroscopy,mutagenesis
更新于2025-09-23 15:23:52
-
Rapid Identification of Functional Pyrrolysyl-tRNA Synthetases via Fluorescence-Activated Cell Sorting
摘要： The orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and their variants have provided powerful tools for expanding the genetic code to allow for engineering of proteins with augmented structure and function not present in Nature. To expedite the discovery of novel pyrrolysyl-tRNA synthetase (PylRS) variants that can charge non-natural amino acids into proteins site-specifically, herein we report a streamlined protocol for rapid construction of the pyrrolysyl-tRNA synthetase library, selection of the functional PylRS mutants using fluorescence-activated cell sorting, and subsequent validation of the selected PylRS mutants through direct expression of the fluorescent protein reporter using a single bacterial strain. We expect that this protocol should be generally applicable to rapid identification of the functional PylRS mutants for charging a wide range of non-natural amino acids into proteins.
关键词： non-natural amino acids,flow cytometry,mutagenesis,amber codon suppression,genetic code expansion
更新于2025-09-23 15:23:52
-
Discovery and Characterization of a Naturally Occurring, Turn-On Yellow Fluorescent Protein Sensor for Chloride
摘要： Fluorescent proteins have been extensively engineered and applied as optical indicators for chloride in a variety of biological contexts. Surprisingly, given the biodiversity of ?uorescent proteins, a naturally occurring chloride sensor has not been reported to date. Here, we present the identi?cation and spectroscopic characterization of the yellow ?uorescent protein from the jelly?sh Phialidium sp. (phiYFP), a rare example of a naturally occurring, excitation ratiometric, and turn-on ?uorescent protein sensor for chloride. Our results show that chloride binding tunes the pKa of the chromophore Y66 and shifts the equilibrium from the ?uorescent phenolate form to the weakly ?uorescent phenol form. The latter likely undergoes excited-state proton transfer to generate a turn-on ?uorescence response that is pH-dependent. Moreover, anion selectivity and mutagenesis in the chloride binding pocket provide additional evidence for the proposed chloride sensing mechanism. Given these properties, we anticipate that phiYFP, with further engineering, could be a new tool for imaging cellular chloride dynamics.
关键词： phiYFP,mutagenesis,chloride sensor,anion selectivity,turn-on fluorescence,pH-dependent,excitation ratiometric,fluorescent proteins
更新于2025-09-23 15:21:21