Redox-Dependent Stability, Protonation, and Reactivity of Cysteine-Bound Heme Proteins

Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys–heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates

Probing a Complex of Cytochromecand Cardiolipin by Magnetic Circular Dichroism Spectroscopy: Implications for the Initial Events in Apoptosis

Oxidation of cardiolipin (CL) by its complex with cytochrome c (cyt c) plays a crucial role in triggering apoptosis. Through a combination of magnetic circular dichroism spectroscopy and potentiometric titrations, we show that both the ferric and ferrous forms of the heme group of a CL:cyt c complex exist as multiple conformers at a physiologically relevant pH of 7.4. For the ferric state, these conformers are His/Lys- and His/OH–-ligated. The ferrous state is predominantly high-spin and, most likely, His/–. Interconversion of the ferric and ferrous conformers is described by a single midpoint potential of -80 ± 9 mV vs SHE. These results suggest that CL oxidation in mitochondria could occur by the reaction of molecular oxygen with the ferrous CL:cyt c complex in addition to the well-described reaction of peroxides with the ferric form

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality

In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function. Site-specific PEGylation at the naturally reactive thiol on human hemoglobin (βCys93) alters hemoglobin oxygen binding affinity and increases its autooxidation rate. In order to avoid this issue, new reactive thiol residues were therefore engineered at sites distant to the heme group and the α/β dimer/dimer interface. The two mutants were βCys93Ala/αAla19Cys and βCys93Ala/βAla13Cys. Gel electrophoresis, size exclusion chromatography and mass spectrometry revealed efficient PEGylation at both αAla19Cys and βAla13Cys, with over 80% of the thiols PEGylated in the case of αAla19Cys. For both mutants there was no significant effect on the oxygen affinity or the cooperativity of oxygen binding. PEGylation at αAla19Cys had the additional benefit of decreasing the rates of autoxidation and heme release, properties that have been considered contributory factors to the adverse clinical side effects exhibited by previous hemoglobin based oxygen carriers. PEGylation at αAla19Cys may therefore be a useful component of future clinical products

T-Lymphocytes Enable Osteoblast Maturation via IL-17F during the Early Phase of Fracture Repair

While it is well known that the presence of lymphocytes and cytokines are important for fracture healing, the exact role of the various cytokines expressed by cells of the immune system on osteoblast biology remains unclear. To study the role of inflammatory cytokines in fracture repair, we studied tibial bone healing in wild-type and Rag1−/− mice. Histological analysis, µCT stereology, biomechanical testing, calcein staining and quantitative RNA gene expression studies were performed on healing tibial fractures. These data provide support for Rag1−/− mice as a model of impaired fracture healing compared to wild-type. Moreover, the pro-inflammatory cytokine, IL-17F, was found to be a key mediator in the cellular response of the immune system in osteogenesis. In vitro studies showed that IL-17F alone stimulated osteoblast maturation. We propose a model in which the Th17 subset of T-lymphocytes produces IL-17F to stimulate bone healing. This is a pivotal link in advancing our current understanding of the molecular and cellular basis of fracture healing, which in turn may aid in optimizing fracture management and in the treatment of impaired bone healing

Infectious diarrhoea in young animals

The aetiology of infectious diarrhoea in young animals, particularly calves, was investigated, using techniques appropriate to the detection of viral, bacterial and protozoal pathogens. Rotavirus was established as of prime significance, and the classic 'white scour' syndrome in calves was usually caused by rotavirus with, on occasion, the simultaneous involvement of coronavirus or Cryptosporidium. Enterotoxigenic Escherichia coli (ETEC) infections were much less common, with under 6% of E. coli isolates possessing K99 fimbriae. Outbreaks of cryptosporidiosis were described for the first time from the U.K. The technique for the detection of the characteristic migration pattern of rotaviral double-stranded RNA segments in silver-stained polyacrylamide gels has proved of especial value in diagnostic and epidemiological investigations.Infection of gnotobiotic lambs with lamb rotavirus produced dullness, inappetance, and diarrhoea, and provided a most useful model for pathogenesis studies. A rapid and extensive infection and defoliation of small intestinal epithelium leading to partial villus atrophy was followed within 2-3 days by a return to apparent morphologic normality. However, the underlying continuing dysfunction of an increased cell turnover rate was demonstrated by metaphase accumulation. Animals with acute enteritis were tolerant to levels of lactose normally found in milk, but their ability to digest and absorb increased oral doses of lactose was impaired. In calves, a concurrent rotavirus infection facilitated intestinal ETEC colonisation beyond the normal age of resistance.Studies on passive immunisation in young lambs demonstrated that protection against rotavirus infection by antibody in the gut lumen was more effective than that provided by circulating antibody. The potential value of this technique was shown in experiments in lambs using rotavirus and immunoglobulin of human origin. Experimental adjuvanted vaccines of inactivated rotavirus given to ewes and cows in pregnancy significantly increased the titre of antibody of IgGl isotype in colostrum and milk. Neonates ingesting these secretions were protected to various degrees against rotavirus infection and diarrhoea. The incorporation of commercially-produced K99 fimbriae from ETEC allowed the successful experimental testing and subsequent field trialling of a vaccine which substantially reduced rotavirus and ETEC diarrhoea problems in the progeny of vaccinated cows. Sero¬ logical variation in rotavirus strains was of potential significance to successful vaccination: atypical rotaviruses with no serological relationship to 'conventional' rotaviruses were identified and characterised serologically and genomically, but occurred too infrequently in calves to present a major clinical problem. Distinct calf rotavirus serotypes that did not confer passive cross protection were identified. Cows produced a heterotypic immune response to all serotypes to which they had pre-existing antibody after vaccination with a single serotype. Passive immunisation may therefore largely overcome the practical problems posed by the existence of many rotavirus serotypes.In the course of this work on neonatal diarrhoea, studies on diagnosis, epidemiology, pathogenesis and biochemistry of other enteropathogens, particularly astrovirus, Cryptosporidium, E. coli and Campylobacters were made. A method for exploiting the genetic control of susceptibility of piglets to adhesion with K88 fimbriae from ETEC was devised and tested