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: 2016-05-12
摘要: With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure-function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus-host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.
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: 2016-05-12
摘要: The oxygen-evolving center (OEC) in photosystem II catalyzes a water splitting reaction. Great efforts have already been made to artificially synthesize the OEC, in order to elucidate the structure-function relationship and the mechanism of the reaction. Now, a new synthetic model makes the best mimic yet of the OEC. This recent study opens up the possibility to study the mechanism of photosystem II and photosynthesis in general for applications in renewable energy and synthetic biology.
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 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-11
摘要: Cation-pi interactions, where protein aromatic residues supply pi systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-pi interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-pi interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-pi interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins.