R-3 Repository :: Browsing by Author "Olson, John S."

Browsing by Author "Olson, John S."

Now showing 1 - 20 of 42

Alkyl isocyanides as transition state analogs for ligand entry and exit in globins
(2008) Blouin, George C.; Olson, John S.
There are two competing models for ligand entry and exit in globins. In the histidine gate model, a channel from the heme iron to the solvent opens by the outward rotation of the His(E7) side chain (7 th residue of the E-helix). In the multiple paths model, ligands diffuse through the protein matrix and exit at multiple points at its surface. Previous workers solved crystal structures to identify channels in myoglobin (Mb) with n-alkyl isocyanides (CNRs), a long flexible ligand that acts as a molecular "Ariadne's thread." When bound to Mb, a CNR points either toward solvent through an opened His(E7) (out conformation) or into the back of the distal pocket (in conformation). To measure the in/out equilibrium in solution, FTIR spectra have been collected for wild-type and mutant MbCNRs. The fraction of CNRs that point in (Fin) is regulated by the distal pocket volume, the freedom of the His(E7) side chain to rotate outward, and an unfavorable hydrophobic effect for CNRs that point outward into solvent. The relative importance of distal histidine flexibility and pocket volume on diatomic ligand binding with Mb has been assessed by correlating F in for bound CNRs with O2 and NO binding parameters. These correlations indicate strongly that: (1) CNRs and diatomic ligands use the histidine gate; (2) the volume of the binding pocket regulates non-covalent ligand capture and covalent bond formation with the iron atom; (3) the "baseball glove" model of ligand binding to Mb applies for all ligands; and (4) CNRs serve as useful transition state analogs for the diatomic ligand binding reactions of all globins.
Alpha-hemoglobin stabilizing protein (AHSP) markedly decreases the redox potential and reactivity of alpha subunits of human HbA with hydrogen peroxide
(The American Society for Biochemistry and Molecular Biology, Inc., 2012) Mollan, Todd L.; Banerjee, Sambuddha; Wu, Gang; Siburt, Claire J.Parker; Tsai, Ah-Lim; Olson, John S.; Weiss, Mitchell J.; Crumbliss, Alvin L.; Alayash, Abdu I.
Background: AHSP modifies redox properties of bound α subunits. Results: Isolated hemoglobin subunits exhibit significantly different redox properties compared to HbA. A significant decrease in the reduction potential of α subunits bound to AHSP results in preferential binding of ferric α. Conclusion: AHSP:α subunit complexes do not participate in ferric-ferryl heme redox cycling. Significance: AHSP binding to α subunits inhibits subunit pseudoperoxidase activity.
Apoglobin stability and ligand movements in mammalian myoglobins
(1998) Scott, Emily Elizabeth; Olson, John S.
The design of recombinant globins as oxygen storage and delivery pharmaceuticals must address two key protein engineering problems, one dealing with the improvement of apoprotein stability and the other with the structural determinants of ligand binding rates. Thirteen naturally occurring apomyoglobins were observed to unfold following the same two transitions as sperm whale apomyoglobin but with variable stabilities. Even with highly similar proteins, it is difficult to identify the individual amino acids and the specific intramolecular noncovalent forces that confer protein stability. Site-directed mutagenesis of apomyoglobins suggests that single point mutations have complex, but generally small effects on the unfolding of apomyoglobin. However, two pig multiple mutants were constructed on the basis of substitution trends in stable mammalian myoglobins, and these genetically engineered proteins had markedly increased overall stabilities. This result suggests that analysis of naturally occurring variants may be the best way to select modifications that inhibit denaturation. Oxygen binding was measured by both conventional and ultrafast laser photolysis techniques for more than 70 myoglobin mutants at 25 different positions. Intramolecular geminate rebinding of oxygen to sperm whale myoglobin occurs on a nanosecond timescale at room temperature and shows two well separated kinetic components, indicating at least two internal sites from which the photodissociated ligands return to the iron. Xenon accelerates the fast reaction but decelerates and diminishes the slower reaction. The rates and proportions of the two components and xenon effects on them vary widely for different mutants and suggest photodissociated ligands occupy xenon site 4 in the distal pocket and xenon site 1 below the plane of the heme. Rebinding from these positions corresponds to the slower geminate phase for oxygen rebinding. Computed bimolecular rate constants for ligand entry suggest that only residues immediately adjacent to the bound ligand and Phe46 are important for ligand movement into and out of the protein. Ligand entry and exit do not appear to occur via transiently connected channels through the interior of the protein. Instead, the kinetic data presented here suggest that ligands enter and escape through a region circumscribed by the distal histidine, Phe46, and the heme propionates.
Chemical and spectral studies of the Lac repressor protein and its trypsin resistant core
(1982) Smith, Ann; Matthews, Kathleen S.; Palmer, Graham; Olson, John S.; Beckingham, Kathleen M.
The core protein produced by mild proteolytic digestion of the lac repressor has been purified on phosphocellulose, The repressor and core proteins were reacted with the sulfhydryl specific reagents, 2-chloromercuri-4-nitrophenol and fluorescein mercuric acetate. Modification of the cysteine residues did not alter the affinity of the proteins for inducer molecules. The operator binding activity of both proteins was unaffected by the reaction with 2-chloromercuri-4-nitrophenol; however, this binding was essentially abolished upon modification with fluorescein mercuric acetate. This loss of operator DNA binding activity in response to modification supports the thesis that determinants for specific DNA binding are located in the core region of the protein. Fluorescence spectral studies on repressor modified with 2-chloromercuri-4-nitrophenol and fluorescein mercuric acetate were performed. The quenching observed upon titration of repressor with either reagent indicates that energy transfer was occurring between the protein tryptophans and the cysteine-conjugated chromophores. Inclusion of dithiothreitol during the titration prevented the labelling of the cysteines; a corresponding decrease in energy transfer was seen. The addition of of inducer produces a blue shift in the repressor emission spectrum, but did not affect the quenching process. The quenching was sensitive to dithiothreitol for the repressor-inducer system as it had been for the repressor protein alone. The spectral behavior of the core protein was essentially identical to that displayed by the repressor.
Correlations between bound n-alkyl isocyanide orientations and pathways for ligand binding in recombinant myoglobins
(1999) Smith, Robert David; Olson, John S.
Subtle differences in Fe-C-O geometry between the recombinant (pH 9, space group P6) and native (pH 7, space group P21) crystal forms of carbon monoxide sperm whale myoglobin are magnified by using n-butyl isocyanide as a probe of distal pocket stereochemistry. Crystal structures of methyl, ethyl, n-propyl and n-butyl isocyanide bound to recombinant wild-type myoglobin were determined and then compared with the corresponding native myoglobin isocyanide structures determined by Ken Johnson (Ph.D. Dissertation, Rice University, 1993). The wild-type structures show all the ligands pointing inward and the distal histidine in the "down" conformation, whereas the native myoglobin n-propyl and n-butyl isocyanide structures show the ligand side chain pointing outward causing upward movement of the distal histidine. In order to examine the causes of these differences, the structures of Mb n-butyl isocyanide in native P21 crystals and recombinant V68F P6 crystals were determined at both pH 7 and 9. The results show that pH and not crystal packing is the key determinant of ligand orientation. Neutral or low pH favors rotation of the distal histidine and outward movement of the side chain of bound n-butyl isocyanide. Four mutant Mb structures were determined to examine the roles of the distal histidine and valine residues in governing ligand orientation. The results demonstrate that His64 (E7) sterically restricts outward movement of the isocyanide side chain since its replacement with alanine (H64A) or an increase in its mobility (F46V) causes the appearance of the "out" ligand conformation. Steric crowding in the protein interior by the V68F mutation is not sufficient to cause the ligand to point outward at pH 9.0. Since the rate and equilibrium constants for myoglobin n-butyl isocyanide show only a small pH dependence, the "in" and "out" conformations appear to have similar free energies. This suggests that ligand movement into the protein interior and outward through the distal histidine gate have similar probabilities which is what has been observed in laser photolysis experiments with oxymyoglobin (Scott, E. E. and Gibson, Q. H. (1997) Biochemistry 36, 11909--11917).
Determination of Ligand Pathways in Globins: Apolar Tunnels Versus Polar Gates
(The American Society for Biochemistry and Molecular Biology, Inc., 2012) Salter, Mallory D.; Blouin, George C.; Soman, Jayashree; Singleton, Eileen Willey; Dewilde, Sylvia; Moens, Luc; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Olson, John S.
Background: O2 pathways in animal hemoglobins and myoglobins are controversial. Results: Ligands enter and exit sperm whale Mb and Cerebratulus lacteus Hb by completely different pathways. Conclusion: Rational mutagenesis mapping can identify ligand migration pathways and provides experimental benchmarks for testing molecular dynamics simulations. Significance: Globins can use either a polar gate or an apolar tunnel for ligand entry.
Determination of Pathways for Oxygen Binding to Human Hemoglobin A
(2011) Birukou, Ivan A.; Olson, John S.
The role of His(E7) in ligand binding to HbA was re-examined in view of discrepancies between early kinetic studies and recent structure determinations. Replacing His(E7) with Gly, Leu, Phe, and Trp causes 20- to 500-fold increases in the rates of O2 dissociation from both subunits, and FfIR spectra reveal a shift in the C-O stretching frequency peak from 1950 cm-1 to -1970 cm-1 for apolar mutations, indicating loss of a positive electrostatic field next to bound ligands. Thus, the native His(E7) side chain forms a strong hydrogen bond (- -8 kJ/mol) with bound 02 in both HbA subunits. Increasing the size of the E7 residue from Gly to Trp monotonically decreases the rate constants for CO, 02 and NO association to HbA. Substituting His(E7) with Trp also slows down opening and closing of the E7 gate. Ligand binding to the Trp(E7) subunits is markedly biphasic due to a competition between very fast recombination to an open conformer and relaxation of the indole side chain to closed or blocked forms. Crystal structures of Hb and Mb Trp(E7) mutants provide structural models for these closed and blocked conformers. In the closed state, the indole side chain fills both the E7 channel and the distal pocket, inhibiting binding to iron from any direction. In the blocked state, Trp(E7) is located in the solvent interface but still blocks entry into the E7 channel. The bimolecular rate constants for CO binding to the closed and blocked states are 0.08 IlMIS- l and 0.7IlM-1S-1, respectively, which are -100 and -lO-fold slower than average wildtype parameter (-7 IlM-1s-1). Filling potential alternative ligand pathways with xenon does not affect the rate or fraction of ligand escape from either HbA subunit. In contrast, reducing the volume of the distal pocket by space-filling mutations at the BlO, Ell and GS positions dramatically affects both geminate recombination and bimolecular ligand binding. Taken together, these results demonstrate that the E7 channel is the major pathway for ligand entry and escape in HbA and that previously proposed ligand migration routes involving Xe cavities are not functionally significant.
Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore
(Public Library of Science, 2012) Ekworomadu, MarCia T.; Poor, Catherine B.; Owens, Cedric P.; Balderas, Miriam A.; Fabian, Marian; Olson, John S.; Murphy, Frank; Balkabasi, Erol; Honsa, Erin S.; He, Chuan; Goulding, Celia W.; Maresso, Anthony W.
To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin.ﾠBacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain ofﾠB. anthracisﾠon hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.
Electrostatic regulation of oxygen and carbon monoxide binding in the alpha and beta subunits of recombinant human hemoglobin
(2004) Schweers, Rachel Leininger; Olson, John S.
The electrostatic theory for ligand discrimination in myoglobin is based on the physiological importance of hydrogen bonding between the distal histidine and bound ligands in myoglobin. A quantitative estimation of this theory in hemoglobin had not been established. A series of 'electrostatic' mutations was created to examine for the existence of electrostatic stabilization of bound O2 in both the alpha and beta subunit of R-state recombinant human hemoglobin. A set of HisE7 to Ala, Leu, and Gln replacements was used to investigate the role of the distal histidine. The aliphatic mutations, AlaE7 and LeuE7, abolish all hydrogen bonding capabilities near bound ligands and cause dramatic increases in ligand association rate constants, particularly for O2. The GlnE7 substitution retains hydrogen bonding capabilities and results in wild-type-like ligand binding behavior. A second set of mutations were created to alter and examine the electrostatic potential in the distal pocket: PheCD4 → Val, ValE11 → Asn, and the double mutant HisE7 → Leu/ValE11 → Asn. The ValCD4 mutation increases the flexibility of the distal histidine side chain, weakening hydrogen bonding interactions with bound ligands. The addition of an asparagine into the distal pocket at position E11 provides a second hydrogen bonding donor in both subunits. The LeuE7/AsnE11 double mutation maintains 'wild-type' electrostatic behavior, due to the compensatory effects of the addition of a hydrogen bond donor at position E11 and removal of one at position E7. Finally, the O2 and CO properties of the double mutant HisE7 → Gln/LeuB10 → Trp were examined as a blood substitute prototype with reduced rates of ligand capture but normal or increased rates of ligand dissociation. This combination of amino acid replacements causes marked decreases in O2 affinity in both subunits and maintains high rates of dissociation, both of which are favorable for efficient O2 transport. The rate of association is decreased by the large size of the Tip side chain and the weakening of hydrogen bonding by the GlnE7 replacement markedly enhances O2 dissociation compared to that measured for the single TrpB10 mutants.
Electrostatic regulation of oxygen binding to the neuronal hemoglobin of Cerebratulus lacteus
(2004) Hale, Angela Dawn; Olson, John S.
The neuronal hemoglobin from Cerebratulus lacteus (CerHb) has an O2 affinity (P50 ≈ 1 muM) similar to mammalian myoglobin (Mb), which are both optimized for oxygen storage and release. Mb has a histidine at the E7 helical position, which provides a strong, stabilizing hydrogen bond to bound O2 and a leucine and valine at the B10 and E11 positions, respectively. In contrast, CerHb has three potential hydrogen bonding donors, tyrosine at B10 and glutamine at E7, called the YQ motif, and an unusual Thr at the E11 position. Invertebrate hemoglobins displaying the YQ motif typically have much higher oxygen affinities (P50 ≤ 0.1 muM) and significantly lower rates of O2 dissociation (≤5 s-1) than Mbs. Using mutagenesis and IR spectroscopy, we have been able to show that the beta-hydroxyl of ThrE11 accepts a proton from the TyrB10 side chain, causing the non-bonded electrons of the phenoxyl group to point toward bound O2. The resultant partial negative charge destabilizes the FeO2 complex, causing the high oxygen dissociation rate constant, kO2, and moderate affinity of the wild-type protein.
Engineering hemoglobins and myoglobins for efficient oxygen transport
(2004) Maillett, David H.; Olson, John S.
Consumption of the intercellular messenger, nitric oxide (NO), by extracellular hemoglobin causes the hypertensive side effect associated with current blood substitute products. Substitution of large aromatic amino acid side chains into the distal cavity of hemoglobin can decrease the rate of this NO scavenging reaction, and mitigate the hypertensive effect. However, such substitutions can also affect O2 binding and release by hemoglobin. To better define the impact of distal pocket mutation on O2 transport, several myoglobin prototypes were tested in an artificial capillary. In addition, a series of recombinant hemoglobins containing phenylalanine and tryptophan substitutions at key locations were characterized to examine how these mutations affect O2 and CO binding. Experiments measuring O2 release and uptake by mutant myoglobins in an artificial capillary demonstrate that delivery is dependent on the affinity of the protein, but that uptake is similar for all of the mutants. This shows that decreases in association rate constants are better tolerated than decreases in dissociation rate constants, and validates the connection between the equilibrium constant for O2 binding, and the physiological transport function of hemoglobin. Placement of Phe and Trp at positions B10, E11 and G8 within the distal pocket decreases the rate at which ligands can gain entry to the active site, which was expected based on similar behavior in myoglobin mutants. The B10 mutants have direct steric and electrostatic interactions with the ligand, seen in the 2000-fold decrease in rate of O2 association for the beta(Trp(B10)) and 60-fold decrease in the O2 dissociation rate constant for alpha(Phe(B10)) subunits. Substitution at position E11 increases O2 and CO affinity due to removal of the naturally occurring gamma2CH3 group of Val(E 11). The E11 mutants have less dramatic effects in hemoglobin than in myoglobin due to the absence of 'extra' volume in the back of the distal cavity in either hemoglobin subunit. Steric crowding by position G8 mutants decreases the rate and extent of ligand capture, particularly in beta subunits. In general, ideas developed for ligand binding in myoglobin translate well to hemoglobin, but structural details of each subunit can magnify or diminish the effect of the mutation.
Enhancing recombinant hemoglobin production by co-expression with alpha hemoglobin stabilizing protein
(2010-01-05) Olson, John S.; Weiss, Mitchell J.; Rice University; Children's Hospital of Philadelphia; United States Patent and Trademark Office
The present disclosure relates to the use of AHSP to stabilize the α subunit of rHb. AHSP may be co-expressed with the hemoglobin genes. AHSP stabilization may be used to increase the production of intact rHb in various systems, such as E. coli, other microorganisms, or animal erythroid cells. This intact rHb may then be used as part of a blood substitute product.
Enhancing Stability and Expression of Recombinant Human Hemoglobin in E. Coli: Progress in the Development of a Recombinant HBOC Source
(2008) Graves, Philip Edwar; Olson, John S.; MacKenzie, Kevin; Tao, Yitzi Jane; Wagner, Daniel
The commercial feasibility of recombinant human Hb (rHb) as an O2 delivery pharmaceutical is limited by the production yield of holoprotein in E. coli. Currently the production ofrHb is not cost effective for use as a source in the development of third and fourth generation Hb-based oxygen carriers (HBOCs). The major problems appear to be aggregation and degradation of apoglobin at the nominal expression temperatures, 28-37° C, and the limited amount of free heme that is available for holohemoglobin assembly. One approach to solve the first problem is to inhibit apoglobin precipitation by a comparative mutagenesis strategy to improve apoglobin stability. aGlyl5 to Ala and P Glyl6 to Ala mutations have been constructed to increase the stability of the A helices of both subunits of adult human hemoglobin (HbA), based on comparison with the sequences of the more stable sperm whale hemoglobin subunits. Human fetal hemoglobin is also known to be more stable than HbA, and comparisons between human P and y (fetal Hb) chains indicate several substitutions that stabilize the aiPi interface, one of which, pHisllb to lie, increases resistance to denaturation and enhances expression in E. coli. These favorable effects of enhanced globin stability can be augmented by co-expression of bacterial membrane heme transport systems to increase the rate and extent of heme uptake through the bacterial cell membranes. The combination of increased apoglobin stability and active heme transport may enhance holohemoglobin production to levels that may make rHb a plausible starting material for all extracellular Hb-based oxygen carriers.
Evolutionary Trajectories to Daptomycin Resistance in Enterococcus faecalis
(2013-11-18) Miller, Corwin; Olson, John S.; Shamoo, Yousif; Bennett, Matthew R.; Rudolf, Volker H. W.; Tao, Yizhi Jane
With increasing amounts of hospital-acquired antibiotic resistant infections each year and staggering healthcare costs, there is a clear need for new antimicrobial agents, as well as novel strategies to extend their clinical efficacy. While genomic studies have provided a wealth of information about the alleles associated with adaptation to antibiotics, they do not provide essential information about relative importance of genomic changes, their order of appearance, or potential epistatic relationships between adaptive changes. In this thesis, I have combined experimental evolution, comparative whole genome sequencing, and allelic frequency measurements to study daptomycin (DAP) resistance in the vancomycin resistant clinical pathogen Enterococcus faecalis strain S613. Maintaining cells inside a turbidostat, a single polymorphic culture was grown sustaining both planktonic and non-planktonic (e.g. biofilm) populations in co-culture as the concentration of antibiotic was raised, facilitating the development of more ecological complexity than is typically observed in laboratory evolution. This approach revealed a clear order and hierarchy of genetic changes leading to resistance, the signaling and metabolic pathways responsible, and the relative importance of these mutations to the evolution of DAP resistance. Genetic and phenotypic comparisons between resistant isolates also identified convergent evolutionary trajectories, suggesting a common biochemical mechanism of resistance. Despite the relative ecological simplicity of this approach compared to the complexity of the human body, I show that experimental evolution can be used to rapidly identify clinically relevant adaptive molecular pathways and new targets for drug design in pathogens.
Expression, Folding, and Assembly Mechanisms of Monomeric Myoglobins and Oligomeric Hemoglobins
(2017-11-16) Mohan Dass, Premila P; Phillips, George N.; Olson, John S.
Despite differing in quaternary structure and protein sequence, mammalian myoglobins and hemoglobins share similar overall globin folds and nearly identical active site structures. The folding mechanism for monomeric apomyoglobin is a well-characterized 2-step pathway involving a molten globule intermediate containing an unfolded heme pocket. Holomyoglobin assembly involves reversible hemin binding to both the molten globule and fully folded apomyoglobin. A wheat germ based cell-free expression assay was developed to show that production levels of folded holomyoglobins correlate quantitatively with their overall apomyoglobin stability constants. Higher cell-free expression levels were also observed for myoglobin mutants with heme cavity filling mutations that significantly increase apomyoglobin stability at the expense of hemin binding affinity. The new in vitro results are consistent with previous observations of myoglobin expression in animal muscle cells and E. coli, all of which demonstrate that apomyoglobin stability is the key determinant of holoprotein expression. In contrast to myoglobin, the individual α and β apoglobin subunits of adult human hemoglobin A (HbA) are extremely unstable, despite being structurally similar to apomyoglobin. GdnHCl induced unfolding curves were measured for human apo- and holo- HbA, fetal hemoglobin, and recombinant hemoglobins with either heme cavity filling apolar mutations or a genetically crosslinked di-α subunit. A mathematical model for hemoglobin tetramer assembly was developed, starting with the mechanism for apohemoglobin folding and adding heme binding steps for each of the different apoprotein states. The unfolding pathway for the heterodimeric apohemoglobin is a 4-step, 5-state mechanism. The first step involves unfolding of the heme pockets to form a heterodimeric molten globule intermediate. This intermediate dissociates into mostly unfolded monomers that then either interact transiently or undergo complete unfolding. Reversible hemin binding to the folded αβ apoHb dimer facilitates formation of the tetrameric α1β2 interfaces, promoting the final assembly of the HbA tetramer. Both the experimental studies and mathematical modeling of hemoglobin assembly provide the framework for understanding human hemoglobinopathies arising from globin misfolding and for enhancing the production yields of heme proteins in bacterial and eukaryotic expression systems.
Hemoglobin mutants that reduce heme loss
(2004-11-02) Olson, John S.; Whitaker, Timothy L.; Hargrove, Mark S.; Rice University; United States Patent and Trademark Office
The present invention relates to methods of reducing heme loss in hemoglobins to produce stability and improve expression yield of hemoglobins, particularly recombinant hemoglobins. Such methods are accomplished by introducing mutations in the alpha or beta subunits of hemoglobins to increase heme affinity. The present invention further relates to novel mutations that reduce such heme loss.
Hemoglobin mutants that reduce heme loss
(2000-09-05) Olson, John S.; Whitaker, Timothy L.; Hargrove, Mark S.; Rice University; United States Patent and Trademark Office
The present invention relates to methods of reducing heme loss in hemoglobins to produce stability and improve expression yield of hemoglobins, particularly recombinant hemoglobins. Such methods are accomplished by introducing mutations in the alpha or beta subunits of hemoglobins to increase heme affinity. The present invention further relates to novel mutations that reduce such heme loss.
Hemoglobin mutants with increased soluble expression and/or reduced nitric oxide scavenging
(2006-05-23) Weickert, Michael J.; Glascock, Christopher B.; Mathews, Antony J.; Lemon, Douglas D.; Doherty, Daniel H.; Olson, John S.; Baxter Biotech Technology SARL; Rice University; United States Patent and Trademark Office
The invention relates to novel recombinant hemoglobins having reduced nitric oxide scavenging and/or increased high soluble expression. The invention further relates to methods of increasing the soluble expression of recombinant hemoglobin by adding exogenous hemin in molar excess of the heme binding sites of recombinant hemoglobin.
Hemoglobin mutants with increased soluble expression and/or reduced nitric oxide scavenging
(2002-09-24) Weickert, Michael J.; Glascock, Christopher B.; Mathews, Antony J.; Lemon, Douglas D.; Doherty, Daniel H.; Olson, John S.; Baxter Biotech Technology SARL; Rice University; United States Patent and Trademark Office
High-resolution X-ray structures of myoglobin- and hemoglobin-alkyl isocyanide complexes
(1993) Johnson, Kenneth Alan; Olson, John S.
The structures of sperm whale myoglobin (Mb) and human hemoglobin (Hb) complexed with methyl, ethyl, n-propyl and n-butyl isocyanide (MNC, ENC, nPNC and nBNC, respectively) were determined by X-ray crystallography. The polar isocyano head groups of the alkyl isocyanides (RNC's) have similar affinities for heme iron, whereas the size and stereochemistry of the alkyl groups cause the different RNC ligands to cross varied steric barriers when entering or exiting the protein. Four Mb structures were determined at a resolution $$ 100$\sp\circ$ rotation about its C$\sb\alpha$-C$\sb\beta$ bond. Relatively immobile side chains of amino acids in the heme pocket allowed the alkyl groups of MNC and ENC to lie in the hydrophobic interior of the heme pocket. The larger alkyl groups of nPNC and nBNC projected into the hydrophilic exterior entrance to the heme pocket. Data for the structures of Hb complexed with MNC, ENC, nPNC and nBNC (pH 6.7) were collected to $\sim$2.2A. In $\alpha$ subunits, the alkyl moieties of the ligands lay approximately parallel to the heme plane. In $\beta$ subunits, the alkyl groups lay at about a 45$\sp\circ$ angle from the heme plane. The alkyl groups of the ligands occupied the hydrophobic interior of the heme pocket in both subunits. Little disorder in His-E7 was observed in either subunit. Ligand binding causes greater tertiary structure changes in the $\alpha$ and $\beta$ subunits than in myoglobin. These changes in tertiary structure in response to isocyanide binding appear to cause the $\alpha$ and $\beta$ subunits to have more of a T-state tertiary conformation than does the oxy complex, and may explain the lesser cooperative effect seen in isocyanide binding compared to oxygen binding.

Browsing by Author "Olson, John S."

Results Per Page

Sort Options