RESUMO
Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer-peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.
Assuntos
Peptídeos , Proteínas , Peptídeos/química , Sequência de Aminoácidos , Proteínas/metabolismo , Simulação de Dinâmica Molecular , Ribossomos/metabolismoRESUMO
Rational design of self-assembled DNA nanostructures has become one of the fastest-growing research areas in molecular science. Particular attention is focused on the development of dynamic DNA nanodevices whose configuration and function are regulated by specific chemical inputs. Herein, we demonstrate the concept of metal-mediated base-pair switching to induce inter- and intramolecular DNA strand displacement in a metal-responsive manner. The 5-hydroxyuracil (UOH) nucleobase is employed as a metal-responsive unit, forming both a hydrogen-bonded UOH-A base pair and a metal-mediated UOH-GdIII-UOH base pair. Metal-mediated strand displacement reactions are demonstrated under isothermal conditions based on the base-pair switching between UOH-A and UOH-GdIII-UOH. Furthermore, metal-responsive DNA tweezers and allosteric DNAzymes are developed as typical models for DNA nanodevices simply by incorporating UOH bases into the sequence. The metal-mediated base-pair switching will become a versatile strategy for constructing stimuli-responsive DNA nanostructures, expanding the scope of dynamic DNA nanotechnology.
Assuntos
DNA Catalítico , DNA , Pareamento de Bases , Hidrogênio , MetaisRESUMO
Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here, we develop a method to rapidly deplete tubulin glutamylation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.
Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Cílios/ultraestrutura , Genes Reporter , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog/genética , Cinesinas , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Células NIH 3T3 , Imagem Óptica , Transdução de Sinais , Tubulina (Proteína)/genética , Tirosina/metabolismoRESUMO
We developed an ultrasound-chemical hybrid tool to precisely manipulate cellular activities. A focused ultrasound coupled with gas-filled microbubbles was used to rapidly trigger the influx of membrane-impermeable chemical dimerizers into living cells to regulate protein dimerization and location without inducing noticeable toxicity. With this system, we demonstrated the successful modulation of phospholipid metabolism triggered by a short pulse of ultrasound exposure. Our technique offers a powerful and versatile tool for using ultrasound to spatiotemporally manipulate the cellular physiology in living cells.