RESUMO
Hemoglobin-based blood substitutes are one of the options available to derive a resuscitating fluid taking into account clinical and physiological demands. In this paper we investigated a novel protein, Hb(alphaalpha,betabeta) obtained as a combination of two homodimers alpha(2) and beta(2) both derived from a fusion gene containing two alfa chains or two beta chains, each respectively coupled via a specific linker. The construct here described is thus a novel heterodimeric hemoglobin carrying four heme groups. The protein cannot dissociate into dimers, as demonstrated by its absence of reactivity versus haptoglobin, and is expected to have a relatively long circulating half-life. The modification does not increase the autoxidation rate, but increases the oxygen affinity, due to a destabilization of the T quaternary state. Characterization of the biochemical properties of this protein in comparison with HbA is reported.
Assuntos
Substitutos Sanguíneos/uso terapêutico , Hemoglobina A/metabolismo , Hemoglobinas/genética , Oxiemoglobinas/uso terapêutico , Automação , Sequência de Bases , Clonagem Molecular , Primers do DNA , Dimerização , Expressão Gênica , Hemoglobina A/genética , Hemoglobina A/uso terapêutico , Hemoglobinas/química , Hemoglobinas/metabolismo , Hemoglobinas/uso terapêutico , Humanos , Cinética , Oxigênio/sangue , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêuticoRESUMO
Studying transcription machinery assembly in vitro is challenging because of long intrinsically disordered regions present within the multi-modular transcription factors. One example is alcohol dehydrogenase repressor 1 (Adr1p) from fermenting yeast, responsible for the metabolic switch from glucose to ethanol. The role of each individual transcription activation domain (TAD) has been previously studied, but their interplay and their roles in enhancing the stability of the protein is not known. In this work, we designed five unique miniAdr1 constructs containing either TADs I-II-III or TAD I and III, connected by linkers of different sizes and compositions. We demonstrated that miniAdr1-BL, containing only PAR-TAD I+III with a basic linker (BL), binds the cognate DNA sequence, located in the promoter of the ADH2 (alcohol dehydrogenase 2) gene, and is necessary to stabilize the heterologous expression. In fact, we found that the sequence of the linker between TAD I and III affected the solubility of free miniAdr1 proteins, as well as the stability of their complexes with DNA. miniAdr1-BL is the stable unit able to recognize ADH2in vitro, and hence it is a promising tool for future studies on nucleosomal DNA binding and transcription machinery assembly in vitro.
Assuntos
Álcool Desidrogenase/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Engenharia de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Sítios de Ligação , Clonagem Molecular , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
After over a century of extensive research, hemoglobin has become the prototype of allosteric and cooperative proteins. Its molecular structure, known in great detail, has allowed the design of hundreds of site directed mutations, aimed at interfering with its function, and thus at testing our hypotheses on the molecular mechanisms of allostery. The wealth of information thus obtained is difficult to read except for specialists, not only because it makes use of many different technical approaches, but also because of its intrinsically patchy nature. Moreover, several researchers have tried to assign specific roles to segments of the polypeptide chains, rather than to single residues, and have tested their hypotheses by multiple point mutations or by complete replacement with the homologous segment from a different hemoglobin to produce chimeric macromolecules. This approach is in great need of a revision since putative functionally relevant segments partially overlap. This review briefly describes the structure and function of hemoglobin, and analyzes the effect of point mutations, multiple mutations and segment replacement, with special attention to possible biotechnological applications, ranging from pharmacology (Hb solutions as resuscitating fluids and sources of the protein found in hemoglobinopathies for biochemical studies) to bioreactors. Occasional reference is made to site directed mutants of myoglobin, whenever this helps clarifying perplexing results obtained on hemoglobin.
Assuntos
Hemoglobinas/química , Regulação Alostérica , Hemoglobinas/genética , Humanos , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação Puntual , Conformação Proteica , Estrutura Quaternária de ProteínaRESUMO
XendoU is the first endoribonuclease described in higher eukaryotes as being involved in the endonucleolytic processing of intron-encoded small nucleolar RNAs. It is conserved among eukaryotes and its viral homologue is essential in SARS replication and transcription. The large-scale purification and crystallization of recombinant XendoU are reported. The tendency of the recombinant enzyme to aggregate could be reversed upon the addition of chelating agents (EDTA, imidazole): aggregation is a potential drawback when purifying and crystallizing His-tagged proteins, which are widely used, especially in high-throughput structural studies. Purified monodisperse XendoU crystallized in two different space groups: trigonal P3(1)21, diffracting to low resolution, and monoclinic C2, diffracting to higher resolution.