RESUMO
The extent to which thermostability influences the location of protein fragmentation sites that allow retention of function is not known. To evaluate this, we used a novel transposase-based approach to create libraries of vectors that express structurally-related fragments of Bacillus subtilis adenylate kinase (BsAK) and Thermotoga neapolitana adenylate kinase (TnAK) with identical modifications at their termini, and we selected for variants in each library that complement the growth of Escherichia coli with a temperature-sensitive adenylate kinase (AK). Mutants created using the hyperthermophilic TnAK were found to support growth with a higher frequency (44%) than those generated from the mesophilic BsAK (6%), and selected TnAK mutants complemented E. coli growth more strongly than homologous BsAK variants. Sequencing of functional clones from each library also identified a greater dispersion of fragmentation sites within TnAK. Nondisruptive fission sites were observed within the AMP binding and core domains of both AK homologs. However, only TnAK contained sites within the lid domain, which undergoes dynamic fluctuations that are critical for catalysis. These findings implicate the flexible lid domain as having an increased sensitivity to fission events at physiological temperatures. In addition, they provide evidence that comparisons of nondisruptive fission sites in homologous proteins could be useful for finding dynamic regions whose conformational fluctuations are important for function, and they show that the discovery of protein fragments that cooperatively function in mesophiles can be aided by the use of thermophilic enzymes as starting points for protein design.
Assuntos
Adenilato Quinase/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Thermotoga neapolitana/enzimologia , Adenilato Quinase/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Domínio Catalítico , Escherichia coli/enzimologia , Temperatura Alta , Dados de Sequência Molecular , Conformação Proteica , Processamento de Proteína Pós-Traducional , Homologia de Sequência de AminoácidosRESUMO
Se presenta una experiencia de 50 casos de onicocriptosis tratados mediante una técnica quirúrgica mínima en la que se extirpa el borde lateral de la placa ungueal con su correspondiente matriz. La intervención se realiza sin incisión cutánea, lo que permite abreviar el período postoperatorio y ofreció, en la experiencia del autor, resultados funcionales y cosméticos muy satisfactorios
Assuntos
Pé , Unhas Encravadas/cirurgia , ArgentinaRESUMO
Se presenta una experiencia de 50 casos de onicocriptosis tratados mediante una técnica quirúrgica mínima en la que se extirpa el borde lateral de la placa ungueal con su correspondiente matriz. La intervención se realiza sin incisión cutánea, lo que permite abreviar el período postoperatorio y ofreció, en la experiencia del autor, resultados funcionales y cosméticos muy satisfactorios
