RESUMO
The de novo design of peptides and proteins has recently emerged as an approach for investigating protein structure and function. Designed, helical peptides provide model systems for dissecting and quantifying the multiple interactions that stabilize secondary structure formation. De novo design is also useful for exploring the features that specify the stoichiometry and stability of alpha-helical coiled coils and for defining the requirements for folding into structures that resemble native, functional proteins. The design process often occurs in a series of discrete steps. Such steps reflect the hierarchy of forces required for stabilizing tertiary structures, beginning with hydrophobic forces and adding more specific interactions as required to achieve a unique, functional protein.
Assuntos
Conformação Proteica , Engenharia de Proteínas , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termodinâmica , Dedos de ZincoRESUMO
The incorporation of unnatural amino acids into proteins by site-specific mutagenesis provides a valuable new methodology for the generation of novel proteins that possess unique structural and functional features.
Assuntos
Mutação , Proteínas/genética , Acilação , Aminoácidos , RNA de Transferência/genética , Supressão Genética , beta-Lactamases/genéticaRESUMO
Beta-turns are a common secondary structure motif found in proteins that play a role in protein folding and stability and participate in molecular recognition interactions. Somatostatin, a peptide hormone possessing a variety of therapeutically-interesting biological activities, contains a beta-turn in its bioactive conformation. The beta-turn and biological activities of somatostatin have been succesfully mimicked in cyclic hexapeptide analogues. Two novel, structured, non-peptidic molecules were developed that are capable of holding the bioactive tetrapeptide sequence of somatostatin analogues in a beta-turn conformation, as measured by somatostatin receptor (SSTR) binding. Template-constrained cyclic peptides in which the ends of the -Tyr-D-Trp-Lys-Val-tetrapeptide were linked by scaffolds based on either an N,N'-dimethyl-N,N'-diphenylurea or a substituted biphenyl system (DJS631 and DJS811, respectively), bound selectively to mouse SSTR2B and rat and human SSTR5 with affinities as high as 1 nM. DJS811, at a dose of 3 mg/kg/day, was shown in a mouse Matrigel model to inhibit angiogenesis to a level of 79%. The development of structured turn scaffolds allows beta-turn sequences to be contained in the context of a compact structure, with less peptidic nature and potentially greater bioavailability than cyclic hexapeptides. These systems can be used to study the determinants of beta-turn formation, as well as to probe the importance of turn sequences occurring in molecular recognition interactions. The antiangiogenic activity of DJS811 suggests that it may have antitumor activity as well. In addition, because SSTR2 is overexpressed on many types of tumors, DJS631 and DJS811 may be useful in the development of agents for tumor imaging or the radiotherapy of cancer.
Assuntos
Peptídeos Cíclicos , Peptídeos Cíclicos/metabolismo , Somatostatina/análogos & derivados , Inibidores da Angiogênese/metabolismo , Animais , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Animais , Mimetismo Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/metabolismo , Compostos de Fenilureia/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Ratos , Receptores de Somatostatina/metabolismo , Relação Estrutura-Atividade , TermodinâmicaRESUMO
An amber suppressor tRNA was prepared in vitro by runoff transcription with T7 RNA polymerase. Both full-length tRNA and truncated tRNA lacking the 3' terminal pCpA from the acceptor stem could be synthesized from the same DNA template. Truncated runoff suppressor tRNA could be enzymatically ligated to phenylalanyl-pCpA to generate aminoacylated full-length suppressor tRNA (Phe-tRNA(CUA)). Phe-tRNA(CUA) is capable of suppressing an amber (UAG) mutation in vitro with equivalent efficiency as suppressor prepared by anticodon-loop replacement of a naturally-isolated tRNA. The ease of suppressor tRNA preparation using this method, compared to anticodon-loop replacement, greatly facilitates the use of chemically acylated suppressor tRNA's for site-specifically incorporating unnatural amino acids into proteins.