RESUMO
The ability to incorporate non-canonical amino acids (ncAA) using translation offers researchers the ability to extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P). Using this enzyme, we demonstrate ribosomal translation of 11 ncAAs including those with novel side chains, α,α-disubstitutions, and cyclic ß-amino acids.
Assuntos
Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Biossíntese de Proteínas/genética , Engenharia de Proteínas , Valina-tRNA Ligase/metabolismoRESUMO
Due to the lowered pKa of 4-fluorohistidine relative to histidine, peptides and proteins containing this amino acid are potentially endowed with novel properties. We report here the optimized synthesis of 4-fluorohistidine and show that it can efficiently replace histidine in in vitro translation reactions. Moreover, peptides containing 6×-fluorohistidine tags are able to be selectively captured and eluted from nickel resin in the presence of his-tagged protein mixtures.
Assuntos
Histidina/análogos & derivados , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Sequência de Aminoácidos , Histidina/metabolismo , Peptídeos/química , Biossíntese de ProteínasRESUMO
The Rosés and Bosch model for preferential solvation is used to analyze the fluorescence behavior of two PRODAN derivatives in binary solvents with one or two protic components. The preferential solvation results suggest that the excited PRODAN derivatives form two H-bonds. The model allows for determining the characteristics of the singly H-bonded excited states. They show red-shifted fluorescence but relatively little quenching. In contrast, the doubly H-bonded excited states are significantly quenched when the protic solvent is a strong H-bond donor (large SA value). With two protic solvents there is little preferential interaction even though the solvents have very different H-bond-forming ability.
RESUMO
Hydrocarbon stapled peptides are promising therapeutics for inhibition of intracellular protein-protein interactions. Here we develop a new high-throughput strategy for hydrocarbon stapled peptide discovery based on mRNA display of peptides containing α-methyl cysteine and cyclized with m-dibromoxylene. We focus on development of a peptide binder to the HPV16 E2 protein.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Evolução Molecular Direcionada/métodos , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Fatores de Transcrição/metabolismo , Alquilação , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Ciclização , Cisteína/química , Hidrocarbonetos Bromados/química , Biblioteca de Peptídeos , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/químicaRESUMO
The ability to introduce non-canonical amino acids into peptides and proteins is facilitated by working within in vitro translation systems. Non-canonical amino acids can be introduced into these systems using sense codon reprogramming, stop codon suppression, and by breaking codon degeneracy. Here, we review how these techniques have been used to create proteins with novel properties and how they facilitate sophisticated studies of protein function. We also discuss how researchers are using in vitro translation experiments with non-canonical amino acids to explore the tolerance of the translation apparatus to artificial building blocks. Finally, we give several examples of how non-canonical amino acids can be combined with mRNA-displayed peptide libraries for the creation of protease-stable, macrocyclic peptide libraries for ligand discovery.
Assuntos
Código Genético , Biblioteca de Peptídeos , Peptídeos/genética , Animais , Códon/genética , Descoberta de Drogas/métodos , Humanos , Ligantes , Compostos Macrocíclicos/química , Peptídeos/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Biossíntese de Proteínas , Engenharia de Proteínas/métodos , Proteínas/genéticaRESUMO
Highly constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly constrained peptides are challenging to prepare. Here, we present a method which utilizes two robust, orthogonal chemical steps to create highly constrained bicyclic peptide libraries. This technology was optimized to be compatible with in vitro selections by mRNA display. We performed side-by-side monocyclic and bicyclic selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nanomolar affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance.