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

Comparison of the oxidative reactivity of recombinant fetal and adult human hemoglobin: implications for the design of hemoglobin-based oxygen carriers.

Hemoglobin based oxygen carriers (HBOCs) have been engineered to replace or augment the oxygen carrying capacity of erythrocytes. However, clinical results have generally been disappointing due, in part due to the intrinsic oxidative toxicity of hemoglobin. The most common HBOC starting material is adult human or bovine hemoglobin. However, it has been suggested that fetal hemoglobin may offer advantages due to decreased oxidative reactivity. Large scale manufacturing of a HBOC will likely ultimately require recombinant sources of human proteins. We therefore directly compared the functional properties and oxidative reactivity of recombinant fetal (rHbF) and recombinant adult (rHbA) hemoglobin. rHbA and rHbF produced similar yields of purified functional protein. No differences were seen in the two proteins in: autoxidation rate; the rate of hydrogen peroxide reaction; NO scavenging dioxygenase activity; and the NO producing nitrite reductase activity. The rHbF protein was: less damaged by low levels of hydrogen peroxide; less damaging when added to human umbilical vein endothelial cells (HUVEC) in the ferric form; and had a slower rate of intrinsic heme loss. The rHbA protein was: more readily reducible by plasma antioxidants such as ascorbate in both the reactive ferryl and ferric states; less readily damaged by lipid peroxides; and less damaging to phosphatidylcholine liposomes. In conclusion in terms of oxidative reactivity there are advantages and disadvantages to the use of recombinant adult or fetal Hb as the basis for an effective HBOC

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Interactions of mitochondrial cytochrome c with phospholipids

Mitochondrial cytochrome c (cyt c) associates with the phospholipid cardiolipin (Cl) through a combination of electrostatic and hydrophobic interactions. The latter occurs by insertion of an acyl chain into cyt c, resulting in the dissociation of the axial Met-80 haem-iron ligand resulting in a five coordinate cyt c/Cl complex that exhibits peroxidatic properties leading to peroxidation of Cl and dissociation of the complex. These events are considered to be pre-apoptotic and culminate with release of cyt c from the mitochondria into the cytoplasm. Two distinct surface regions on cyt c have been suggested to mediate Cl acyl chain insertion and Chapters 2 and 3 of this thesis report the results of probing one of these regions by constructing a series of alanine mutants in yeast iso-l-cyt c aimed at disrupting a surface cleft formed between residues 67-71 and 82-85. The physicochemical properties, peroxidase activity, Cl binding, and kinetics of carbon monoxide (CO) binding to the ferrous cyt c/Cl complex have been assessed for each individual mutant. The findings reveal that the majority of mutants are capable of binding Cl in the same apparent stoichiometry as the wild-type protein. Mutation of the species conserved Arg-91 residue, that anchors the cleft, results in the greatest changes to physicochemical properties of the protein leading to a change in the Cl binding ratio required to effect structural changes and to the ligand-exchange properties of the ferrous cyt c/CL complex. The second part of this thesis, Chapters 4 and 5, switches focus to human cyt c. A recently discovered natural mutant of human cyt c - G41S, was found to have enhanced apoptotic activity in megakaryocytes leading to thrombocytopenia. An expression construct for WT human cyt c was prepared enabling the G41S mutation to be made and studied. Peroxidase activity in the presence and absence of three phospholipids (TOCl, DOPS and DOPG) was studied and revealed enhanced activity for the G41S mutant in the presence of TOCL. To correlate a mechanism of peroxidase activity in the human cyt c/lipid complex an extensive EPR study was carried out which identifies the spin state changes of haem-iron upon peroxidation of cyt c/lipid complexes. Furthermore, the EPR study identifies that a Tvr radical is formed during peroxide turnover by the cyt c/lipid complex. Identification of which of the five Tyr residues in human cyt c the radical resides has been aided by the crystal structure of human cyt c with Tyr- 46 or Tyr-48 being identified. Further work is required to confirm which of the two Tyr residues act as the radical site.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Structural and kinetic studies of imidazole binding to two members of the cytochrome c 6 family reveal an important role for a conserved heme pocket residue

The amino acid at position 51 in the cytochrome c 6 family is responsible for modulating over 100 mV of heme midpoint redox potential. As part of the present work, the X-ray structure of the imidazole adduct of the photosynthetic cytochrome c 6 Q51V variant from Phormidium laminosum has been determined. The structure reveals the axial Met ligand is dissociated from the heme iron but remains inside the heme pocket and the Ω-loop housing the Met ligand is stabilized through polar interactions with the imidazole and heme propionate-6. The latter is possible owing to a 180° rotation of both heme propionates upon imidazole binding. From equilibrium and kinetic studies, a Val residue at position 51 increases the stability of the Fe-S(Met) interaction and also affects the dynamics associated with imidazole binding. In this respect, the k obs for imidazole binding to Arabidopsis thaliana cytochrome c 6A, which has a Val at the position equivalent to position 51 in photosynthetic cytochrome c 6, was found to be independent of imidazole concentration, indicating that the binding process is limited by the Met dissociation rate constant (about 1 s -1). For the cytochrome c 6 Q51V variant, imidazole binding was suppressed in comparison with the wild-type protein and the V52Q variant of cytochrome c 6A was found to bind imidazole readily. We conclude that the residue type at position 51/52 in the cytochrome c 6 family is additionally responsible for tuning the stability of the heme iron-Met bond and the dynamic properties of the ferric protein fold associated with endogenous ligand binding. © 2011 SBIC

Ascorbate peroxidase activity of cytochrome<i>c</i>

The peroxidase-type reactivity of cytochrome c is proposed to play a role in free radical production and/or apoptosis. This study describes cytochrome c catalysis of peroxide consumption by ascorbate. Under conditions where the sixth coordination position at the cytochrome c heme iron becomes more accessible for exogenous ligands (by carboxymethylation, cardiolipin addition or by partial denaturation with guanidinium hydrochloride) this peroxidase activity is enhanced. A reaction intermediate is detected by stopped-flow UV-vis spectroscopy upon reaction of guanidine-treated cytochrome c with peroxide, which resembles the spectrum of globin Compound II species and is thus proposed to be a ferryl species. The ability of physiological levels of ascorbate (10μM) to interact with this species may have implications for mechanisms of cell signalling or damage that are based on cytochrome c/peroxide interactions. © 2011 Informa UK, Ltd

Backbone resonance assignments of ferric human cytochrome c and the pro-apoptotic G41S mutant in the ferric and ferrous states

Human cytochrome c is a multi-functional protein with key roles in both the mitochondrial electron transfer chain and in apoptosis. In the latter, a complex formed between the mitochondrial phospholipid cardiolipin and cytochrome c is crucial for instigating the release of pro-apoptotic factors, including cytochrome c, from the mitochondrion into the cytosol. The G41S mutant of human cytochrome c is the only known disease-related variant of cytochrome c and causes increased apoptotic activity in patients with autosomal dominant thrombocytopenia. NMR spectroscopy can be used to investigate the interaction of human cytochrome c with cardiolipin and the structural and dynamic factors, which may contribute to enhanced apoptotic activity for the G41S mutant. We present here essentially full backbone amide resonance assignments for ferric human cytochrome c (98 %) as well as assignments of both the ferric (92 %) and ferrous (95 %) forms of the G41S mutant. Backbone amide chemical shift differences between the wild type and G41S mutant in the ferric state reveals significant changes around the mutation site, with many other amides also affected. This suggests the possibility of increased dynamics and/or a change in the paramagnetic susceptibility tensor of the G41S mutant relative to the wild type protein

Structural Dissection of Two Redox Proteins from the Shipworm Symbiont Teredinibacter turnerae

The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria

An investigation into a cardiolipin acyl chain insertion site in cytochrome c

Mitochondrial cytochrome c associates with the phosphoplipid cardiolipin (CL) through a combination of electrostatic and hydrophobic interactions. The latter occurs by insertion into cytochrome c of an acyl chain, resulting in the dissociation of the axial Met-80 heme-iron ligand. The resulting five coordinate cytochrome c/CL complex has peroxidatic properties leading to peroxidation of CL and dissociation of the complex. These events are considered to be pre-apoptotic and culminate with release of cytochrome c from the mitochondria into the cytoplasm. Two distinct surface regions on cytochrome c have been suggested to mediate CL acyl chain insertion and this study has probed one of these regions. We have constructed a series of alanine mutants aimed at disrupting a surface cleft formed between residues 67-71 and 82-85. The physicochemical properties, peroxidase activity, CL binding, and kinetics of carbon monoxide (CO) binding to the ferrous cytochrome c/CL complex have been assessed for the individual mutants. Our findings reveal that the majority of mutants are capable of binding CL in the same apparent stoichiometry as the wild-type protein, with the extent to which the Met-80 ligand is bound in the ferrous cytochrome c/CL complex being mutant specific at neutral pH. Mutation of the species conserved Arg-91 residue, that anchors the cleft, results in the greatest changes to physicochemical properties of the protein leading to a change in the CL binding ratio required to effect structural changes and to the ligand-exchange properties of the ferrous cytochrome c/CL complex. © 2012 Elsevier B.V. © 2012 Elsevier B.V. All rights reserved