RESUMO
The sequence of octarellin I, the first de novo (beta/alpha)8 polypeptide, was revised according to several criteria, among others the symmetry of the sequence, beta-residue volume and hydrophobicity, and charge distribution. These considerations and the overall conclusions drawn from the first design led to two new sequences, corresponding to octarellins II and III. Octarellin II retains perfect 8-fold symmetry. Octarellin III has the same sequence as octarellin II, except for the beta-strands which exhibit a 4-fold symmetry. The two proteins were produced in Escherichia coli. Infrared and CD spectral analyses of octarellins II and III reveal a high secondary structure content. Non-denaturing gel electrophoresis, molecular sieve chromatography and analytical ultracentrifugation suggest that both of these second-generation artificial polypeptides exist as a mixture of a monomer and a dimer form. Octarellins II and III are at least 10 times more soluble than octarellin I. Urea-induced unfolding followed by fluorescence emission suggests that the tryptophan residues, designed to be buried in the (beta/alpha)8, are indeed packed in the hydrophobic core of both proteins. However, octarellin III displays a higher stability towards urea denaturation, indicating that introducing 4-fold symmetry into the beta-barrel might be important for stability of the overall folding.
Assuntos
Engenharia de Proteínas/métodos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Cromatografia em Gel , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas Fúngicas/química , Genes Sintéticos , Dados de Sequência Molecular , Peptídeos/genética , Desnaturação Proteica , Proteínas de Protozoários/química , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Ultracentrifugação , Ureia/farmacologiaRESUMO
In order to investigate how structural modifications interfere with protein stability, we modified a (beta alpha)-unit in E.coli triosephosphate isomerase (TIM), a typical (beta alpha)-barrel protein, assuming that the pseudosymmetrical beta-barrel can be divided into eight successive loop/beta-strand/loop/alpha-helix motifs. We replaced the eighth (beta alpha)-unit of E.coli TIM with the corresponding chicken (beta alpha)-unit. The substitution, involving the replacement of 10 of the 23 residues of this (beta alpha)-unit, was evaluated first by modelling, then experimentally. Modelling by homology suggests how the amino acid replacements might be accommodated in the hybrid E.coli/chicken TIM (ETIM8CHI). Both natural and hybrid recombinant TIMs, overproduced in E.coli, were purified to homogeneity and characterized as to their stability and kinetics. Our kinetic studies show that the modification performed here leads to an active enzyme. The stability studies indicate that the stability of ETIM8CHI is comparable to that of the wild type TIM.
Assuntos
Triose-Fosfato Isomerase/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas/genética , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismoRESUMO
Recombinant tissue inhibitor of metalloproteinases (TIMP-1) and a truncated version containing only the three N-terminal loops, delta 127-184TIMP, have been expressed in myeloma cells and purified by affinity chromatography and gel filtration. delta 127-184TIMP was found to exist as two main glycosylation variants of molecular mass 24 kD and 19.5 kDa and an unglycosylated form of 13 kDa. All forms of the truncated inhibitor were able to inhibit and form complexes with active forms of the matrix metalloproteinases, indicating that the major structural features for specific interaction with these enzymes resides in these three loops. Stable binding of delta 127-184TIMP to pro 95-kDa gelatinase was not demonstrable under the conditions for binding of full-length TIMP-1.
