RESUMEN
One might think that after over 100 years of study we now know all there is to know about Hemoglobin and its function. However, the purpose of this review is to outline that this fascinating protein has still much to say in the field of biological modulation. Hence, we like to focus on a number of parallel functions of hemoglobin besides its basic function of oxygen transport. Among these we like to recall the following main functions: a) modulation of erythrocyte metabolism; b) Heme oxidation and erythrocytes senescence; c) resistance to malaria; d) molecular heat transducer e) Enzymatic activity; f) Hemorphins, carbon monoxide and nitric oxide.
Asunto(s)
Hemo , Hemoglobinas , Monóxido de Carbono/metabolismo , Eritrocitos/metabolismo , Hemo/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Óxido Nítrico/metabolismo , OxígenoRESUMEN
Basic research on hemoglobin has been essential for understanding the origin and treatment of many hematological disorders due to abnormal hemoglobins. The most important of the hemoglobinopathies is sickle cell disease - Linus Pauling's "molecular disease" that gave birth to molecular medicine. In this review, I will describe the contributions of basic biophysical research on normal and sickle cell hemoglobin (HbS) to understanding the molecular pathogenesis of the disease and providing the conceptual basis for the various approaches to drug therapy that target HbS polymerization. Most prominent among these are the experimental results on the solubility of HbS as a function of oxygen saturation explained by the allosteric model of Monod, Wyman, and Changeux and the Gill-Wyman thermodynamic linkage relation between solubility and oxygen binding, the solubility of mixtures of HbS with normal or fetal hemoglobin explained by Minton's thermodynamic model, and the highly unusual kinetics of HbS polymerization explained by a novel double nucleation mechanism that also accounts for the aggregation kinetics of the Alzheimer's peptide. The HbS polymerization kinetics are of great importance to understanding the pathophysiology and clinical course, as well as guiding drug development for treating this common and severe disease. The article focuses primarily on experimental and theoretical results from my lab, so it is not a comprehensive review of the subject.
Asunto(s)
Anemia de Células Falciformes , Hemoglobina Falciforme , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/metabolismo , Hemoglobina Falciforme/química , Hemoglobina Falciforme/metabolismo , Hemoglobina Falciforme/uso terapéutico , Hemoglobinas/química , Humanos , Cinética , TermodinámicaRESUMEN
Hemoglobin (Hb) is the prototypical example of a cooperative protein. Cooperativity of Hb is largely accounted for by the oxygen-linked allosteric interconversion between the T and R states/structures. Allostery is such a powerful explanation of Hb cooperativity that the possibility of cooperative events occurring within each allosteric conformation, in the absence of any quaternary structural change has usually been overlooked, and actually experiments specifically aimed at detecting nonallosteric cooperativity have usually failed to do so. However there are strong, but often neglected, theoretical reasons pointing to the presence of nonallosteric cooperativity under common experimental conditions, that have recently raised new interest and have been thoroughly re-investigated. Non-allosteric cooperativity within T state Hb has often been invoked to describe puzzling experimental data, either as an intrinsic property of the macromolecule or as a consequence of the binding of non-heme ligands. Few convincing pieces of evidence exist for the former hypothesis, whereas very strong proofs are available for effector-induced non-allosteric cooperativity in hemoglobin. Moreover, non-allosteric cooperativity in THb may explain some hitherto puzzling findings, e.g. the bi-exponential O2 release from THb observed by Q.H. Gibson in oxygen pulse experiments, the invariance of L4 found by K. Imai, the cooperative ligand binding by crystals of T state Hb Rotschild, and, possibly, the cooperativity observed in at least some mixed metal hybrid Hbs.
Asunto(s)
Hemoglobinas/metabolismo , Modelos Moleculares , Regulación Alostérica , Hemoglobinas/química , Humanos , Ligandos , Oxígeno/metabolismo , Unión Proteica , Conformación Proteica , TermodinámicaRESUMEN
Class II bifunctional biotin protein ligases (BirA), which catalyze post-translational biotinylation and repress transcription initiation, are broadly distributed in eubacteria and archaea. However, it is unclear if these proteins all share the same molecular mechanism of transcription regulation. In Escherichia coli the corepressor biotinoyl-5'-AMP (bio-5'-AMP), which is also the intermediate in biotin transfer, promotes operator binding and resulting transcription repression by enhancing BirA dimerization. Like E. coli BirA (EcBirA), Staphylococcus aureus, and Bacillus subtilis BirA (Sa and BsBirA) repress transcription in vivo in a biotin-dependent manner. In this work, sedimentation equilibrium measurements were performed to investigate the molecular basis of this biotin-responsive transcription regulation. The results reveal that, as observed for EcBirA, Sa, and BsBirA dimerization reactions are significantly enhanced by bio-5'-AMP binding. Thus, the molecular mechanism of the Biotin Regulatory System is conserved in the biotin repressors from these three organisms.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Bacillus subtilis/química , Biotina/análogos & derivados , Ligasas de Carbono-Nitrógeno/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas Represoras/química , Staphylococcus aureus/química , Transcripción Genética , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Sitios de Unión , Biotina/química , Biotina/metabolismo , Biotinilación , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Clonación Molecular , Secuencia Conservada , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , TermodinámicaRESUMEN
Protein aggregation is a major issue affecting the long-term stability of protein preparations. Proteins exist in equilibrium between the native and denatured or partially denatured conformations. Often denatured or partially denatured conformations are prone to aggregate because they expose to solvent the hydrophobic core of the protein. The aggregation of denatured protein gradually shifts the protein equilibrium toward increasing amounts of denatured and ultimately aggregated protein. Recognizing and quantitating the presence of denatured protein and its aggregation at the earliest possible time will bring enormous benefits to the identification and selection of optimal solvent conditions or the engineering of proteins with the best stability/aggregation profile. In this article, a new approach that allows simultaneous determination of structural stability and the amount of denatured and aggregated protein is presented. This approach is based on the analysis of the concentration dependence of the Gibbs energy (ΔG) of protein stability. It is shown that three important quantities can be evaluated simultaneously: (i) the population of denatured protein, (ii) the population of aggregated protein, and (iii) the fraction of denatured protein that is aggregated.
Asunto(s)
Antineoplásicos/química , Anhidrasa Carbónica II/química , Cetuximab/química , Modelos Moleculares , Agregado de Proteínas , Trastuzumab/química , Animales , Arginina/química , Bovinos , Estabilidad de Medicamentos , Estabilidad de Enzimas , Calor/efectos adversos , Indicadores y Reactivos/química , Concentración Osmolar , Agregación Plaquetaria , Conformación Proteica , Desnaturalización Proteica , Estabilidad Proteica , Solubilidad , Termodinámica , Urea/químicaRESUMEN
Binding of ligands, ranging from proteins to ions, to membrane proteins is associated with absorption or release of heat that can be detected by isothermal titration calorimetry (ITC). Such measurements not only provide binding affinities but also afford direct access to thermodynamic parameters of binding--enthalpy, entropy and heat capacity. These parameters can be interpreted in a structural context, allow discrimination between different binding mechanisms and guide drug design. In this review, we introduce advantages and limitations of ITC as a methodology to study molecular interactions of membrane proteins. We further describe case studies where ITC was used to analyze thermodynamic linkage between ions and substrates in ion-coupled transporters. Similar type of linkage analysis will likely be applicable to a wide range of transporters, channels, and receptors.