Modulation of allosteric control and evolution of hemoglobin

Allostery arises when a ligand-induced change in shape of a binding site of a protein is coupled to a tertiary/quaternary conformational change with a consequent modulation of functional properties. The two-state allosteric model of Monod, Wyman and Changeux [J. Mol. Biol. 1965; 12, 88-118] is an elegant and effective theory to account for protein regulation and control. Tetrameric hemoglobin (Hb), the oxygen transporter of all vertebrates, has been for decades the ideal system to test for the validity of the MWC theory. The small ligands affecting Hb's behavior (organic phosphates, protons, bicarbonate) are produced by the red blood cell during metabolism. By binding to specific sites, these messengers make Hb sensing the environment and reacting consequently. HbI and HbIV from trout and human HbA are classical cooperative models, being similar yet different. They share many fundamental features, starting with the globin fold and the quaternary assembly, and reversible cooperative O2 binding. Nevertheless, they differ in ligand affinity, binding of allosteric effectors, and stability of the quaternary assembly. Here, we recollect essential functional properties and correlate them to the tertiary and quaternary structures available in the protein databank to infer on the molecular basis of the evolution of oxygen transporters

Pichia pastoris Fep1 is a [2Fe-2S] protein with a Zn finger that displays an unusual oxygen-dependent role in cluster binding

Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys(2)-Cys(2) type zinc fingers and a set of four conserved cysteines arranged in a Cys-X-5-Cys-X-8-Cys-X-2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster

Studio delle relazioni struttura-funzione in mutnti di emoglobina umana

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits using five single and three double mutants of human Hb. A surprisingly good linear correlation between the change in the free energy of associ- ation of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity. Moreover, we have gained insight into the mechanism of discrimination between gaseous ligands inside the heme binding pocket by functionally and structurally studying three additional double mutants. One of these mutants may be considered as candidate for the synthesis of a possible ``blood substitute'', which should yield an O2 adduct stable to autoxidation and slowly reacting with NO

Visita al Laboratory of Molecular Biology del Medical Research Centre di Cambridge (GB)

Parte finale del post-dottorato per terminare gli studi sull'endocitosi umanas

Preface

The book entitled “Schistosomiasis: Epidemiology, Diagnosis and Treatment", presents a very interesting overview on this tropical parasitic disease in the post-genomic era. It is a collection of 8 chapters written by world-renown health professionals and researchers, highly active in the field. The mainly targeted public is MDs specializing in Tropical Medicine as well as experienced researchers and professionals who are faced with the challenges of this ancient threat to human health

Attivita' di Ricerca presso l'Istituto di Genetica e Biologia Molecolare e Cellulare di Strasburgo (F)

Espressione eterologa in Pichia pastoris di proteasi del parassita umano Schistosoma manson

Corso di Biocristallografia per studenti del Dottorato di Ricerca In Biochimica

Il Dr Evans ha tenuto una serie di Seminari di biocristallografia per gli studenti del Dottorato di Ricerca in Biochimic

Hemoglobin allostery: new views on old players.

Proteins are dynamic molecular machines whose structure and function are modulated by environmental perturbations and natural selection. Allosteric regulation, discovered in 1963 as a novel molecular mechanism of enzymatic adaptation [Monod, Changeux & Jacob (1963). J. Mol. Biol.6, 306-329], seems to be the leit motiv of enzymes and metabolic pathways, enabling fine and quick responses toward external perturbations. Hemoglobin (Hb), the oxygen transporter of all vertebrates, has been for decades the paradigmatic system to test the validity of the conformational selection mechanism, the conceptual innovation introduced by Monod, Wyman and Changeux. We present hereby the results of a comparative analysis of structure, function and thermodynamics of two extensively investigated hemoglobins: human HbA and trout HbI. They represent a unique and challenging comparison to test the general validity of the stereochemical model proposed by Perutz. Indeed both proteins are ideal for the purpose being very similar yet very different. In fact, T-HbI is a low-ligand-affinity cooperative tetrameric Hb, insensitive to all allosteric effectors. This remarkable feature, besides being physiologically sound, supports the stereochemical model, given that the six residues identified in HbA as responsible for the Bohr and the 2,3-di-phosphoglycerate effects are all mutated. Comparison of the three-dimensional structures of HbA and T-HbI allows unveiling the molecular mechanism whereby the latter has a lower O2 affinity. Moreover, the energetic balance sheet shows that the salt bridges breaking upon allosteric quaternary transition are important yet insufficient to account for the free energy of heme-heme interactions in both hemoglobins

The carbon monoxide derivative of human hemoglobin carrying the double mutation LeuB10->Tyr and HisE7->Gln on alpha and beta chains probed by infrared spectroscopy

The fine structural properties of the distal heme pocket have been probed by infrared spectroscopy of ferrous carbon monoxy human hemoglobin mutants carrying the mutations LeuB10-->Tyr and HisE7-->Gln on the alpha, beta, and both chains, respectively. The stretching frequency of iron-bound carbon monoxide occurs as a single broad band around 1943 cm(-1) in both the alpha and the beta mutated chains. Such a frequency value indicates that no direct hydrogen bonding exists between the bound CO molecule and the TyrB10 phenolic oxygen, at variance with other naturally occurring TyrB10, GlnE7 nonvertebrate hemoglobins. The rates of carbon monoxide release have been determined for the first time by a Fourier transform infrared spectroscopy stopped-flow technique that allowed us to single out the heterogeneity in the kinetics of CO release in the alpha and beta chains for the mutated proteins and for native HbA. The rates of CO release are 15- to 20-fold faster for the mutated alpha or beta chains with respect to the native ones consistent with the lack of distal stabilization on the iron-bound CO molecule. The present results demonstrate that residues in key topological positions (namely E7 and B10) for the distal steric control of the iron-bound ligand are not interchangeable among hemoglobins from different species