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1.  Structure of fully liganded Hb ζ2β2 s trapped in a tense conformation 
The crystallographic analysis of fully liganded Hb ζ2β2 s trapped in a tense conformation is reported.
A variant Hb ζ2β2 s that is formed from sickle hemoglobin (Hb S; α2β2 s) by exchanging adult α-globin with embryonic ζ-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb ζ2β2 s inhibits the polymerization of deoxy­genated Hb S in vitro and reverses characteristic features of SCD in vivo in mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (α2β2), Hb ζ2β2 s exhibits atypical properties that include a high oxygen affinity, reduced cooperativity, a weak Bohr effect and blunted 2,3-­diphosphoglycerate allostery. Here, the 1.95 Å resolution crystal structure of human Hb ζ2β2 s that was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb ζ2β2 s displays a central water cavity, a ζ1–βs2 (or ζ2–βs1) interface, intersubunit salt-bridge/hydrogen-bond interactions, C-terminal βHis146 salt-bridge interactions, and a β-cleft, that are highly unusual for a relaxed hemoglobin structure and are more typical of a tense conformation. These quaternary tense-like features contrast with the tertiary relaxed-like conformations of the ζ1βs1 dimer and the CD and FG corners, as well as the overall structures of the heme cavities. This crystallographic study provides insights into the altered oxygen-transport properties of Hb ζ2β2 s and, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.
PMCID: PMC3792644  PMID: 24100324
hemoglobin; tense state; relaxed state; Bohr effect; 2,3-disphosphoglycerate; cooperativity; allostery
3.  Elevated sphingosine-1-phosphate promotes sickling and sickle cell disease progression 
The Journal of Clinical Investigation  2014;124(6):2750-2761.
Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.
PMCID: PMC4089467  PMID: 24837436
4.  Investigating the Magnetic Susceptibility Properties of Fresh Human Blood for Non Invasive Oxygen Saturation Quantification 
Quantification of blood oxygen saturation on the basis of a measurement of its magnetic susceptibility demands knowledge of the difference in volume susceptibility between fully oxygenated and fully deoxygenated blood (Δχdo). However, two very different values of Δχdo are currently in use. In this work we measured Δχdo as well as the susceptibility of oxygenated blood relative to water, Δχoxy, by MR susceptometry in samples of freshly drawn human blood oxygenated to various levels, from 6 to 98% as determined by blood gas analysis. Regression analysis yielded 0.273 ± 0.006 and − 0.008 ± 0.003 ppm (cgs) respectively, for Δχdo and Δχoxy, in excellent agreement with previous work by Spees et al (MRM 2001;45:533–542).
PMCID: PMC3307833  PMID: 22162033
MR oximetry; MR susceptometry; Susceptibility imaging; Blood oxygen saturation
5.  Hb Baden: Structural and functional characterization 
American journal of hematology  2010;85(11):848-852.
Hb Baden (β18Val→Met) is a rare variant hemoglobin that has never been functionally or clinically characterized. We describe a Hb Baden heterozygote who exhibits normal growth and development, as well as age- and gender-appropriate hematological parameters. Surprisingly, in vitro analyses demonstrate that Hb Baden is relatively unstable and exhibits an abnormally high affinity for O2. These properties are likely to affect the physiologies of individuals who inherit the βBaden mutation in trans to a determinant for either a functionally relevant hemoglobin-opathy or a mild thalassemia. The data also provide insights into the function of the AB-segment/A-helix of the β-globin, supporting a structural model in which this poorly understood region serves as a scaffold that fixes the positions of other helices that directly impact β-globin function.
PMCID: PMC3618950  PMID: 20872549
6.  Hemoglobin Lake Tapawingo [α46 (CE4)Phe→Ser; HBA2:c.140T>C]: a New Unstable α Chain Hemoglobin Variant Associated with Low Systemic Arterial Saturation 
Hemoglobin  2011;35(4):411-416.
A new unstable alpha globin variant was detected in a child with hypoxemia and anemia. The child’s mother was found to carry the same mutation. The hemoglobin variant co-eluted with Hb A2 by cation-exchange high performance liquid chromatography (CE-HPLC) and appeared cathodal to Hb A and anodal to Hb F by isoelectric focusing. It represented less than 20% of the total hemoglobin and was unstable by isopropanol testing. Gene sequencing identified a missense mutation in the α2 gene [HBA2:c.140T>C]. Oxygen dissociation and P50 test results were normal.
PMCID: PMC3616395  PMID: 21797707
Hemoglobin (Hb) Lake Tapawingo; α globin variant; unstable hemoglobin (Hb); oxygen saturation
7.  Crystallographic analysis of human hemoglobin elucidates the structural basis of the potent and dual antisickling activity of pyridyl derivatives of vanillin. Corrigendum 
A correction to the paper by Abdulmalik et al. [(2011), Acta Cryst. D67, 920–928].
The affiliation of one of the authors of Abdulmalik et al. (2011) [Acta Cryst. D67, 920–928] is corrected.
PMCID: PMC3337008
hemoglobin; oxygen affinity; sickle-cell disease; polymerization; T state; R2 state; corrigendum
8.  Crystallographic analysis of human hemoglobin elucidates the structural basis of the potent and dual antisickling activity of pyridyl derivatives of vanillin 
Pyridyl derivatives of vanillin increase the fraction of the more soluble oxygenated sickle hemoglobin and/or directly increase the solubility of deoxygenated sickle hemoglobin. Crystallographic analysis reveals the structural basis of the potent and dual antisickling activity of these derivatives.
Vanillin has previously been studied clinically as an antisickling agent to treat sickle-cell disease. In vitro investigations with pyridyl derivatives of vanillin, including INN-312 and INN-298, showed as much as a 90-fold increase in antisickling activity compared with vanillin. The compounds preferentially bind to and modify sickle hemoglobin (Hb S) to increase the affinity of Hb for oxygen. INN-312 also led to a considerable increase in the solubility of deoxygenated Hb S under completely deoxygenated conditions. Crystallographic studies of normal human Hb with INN-312 and INN-298 showed that the compounds form Schiff-base adducts with the N-terminus of the α-subunits to constrain the liganded (or relaxed-state) Hb conformation relative to the unliganded (or tense-state) Hb conformation. Interestingly, while INN-298 binds and directs its meta-positioned pyridine-methoxy moiety (relative to the aldehyde moiety) further down the central water cavity of the protein, that of INN-312, which is ortho to the aldehyde, extends towards the surface of the protein. These studies suggest that these compounds may act to prevent sickling of SS cells by increasing the fraction of the soluble high-affinity Hb S and/or by stereospecific inhibition of deoxygenated Hb S polymerization.
PMCID: PMC3211971  PMID: 22101818
hemoglobin; oxygen affinity; sickle-cell disease; polymerization; T state; R2 state
9.  A Reverse Time-Course Method for Transcriptional Chase Analyses of mRNA Half-Lives in Cultured Cells 
PLoS ONE  2012;7(7):e40827.
Standard methods for assessing mRNA stabilities in intact cells are labor-intensive and can generate half-life (t1/2) measures that are both imprecise and inaccurate. We describe modifications to a conventional tetracycline-conditional transcriptional chase method for analyzing mRNA stability that significantly simplify its conduct, while generating highly reproducible and accurate t1/2 values. The revised method–which is conducted as a reverse time course, and which accounts for interval expansion in the number of cultured cells–is validated for the analyses of mRNAs with both short and long half-lives. This approach facilitates accurate assessment of mRNA metabolism, providing a user-friendly tool for detailed investigations into their structures and functions, as well as the processes that contribute to their post-transcriptional regulation.
PMCID: PMC3396636  PMID: 22808270
10.  An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis 
The Journal of Clinical Investigation  2007;117(7):1856-1865.
Erythrocyte precursors produce abundant α- and β-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. αHb-stabilizing protein (AHSP) binds free α subunits reversibly to maintain their structure and limit their ability to generate reactive oxygen species. Accordingly, loss of AHSP aggravates the toxicity of excessive free α-globin caused by β-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free α-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 α-globin genes (genotype Ahsp–/–α-globin*α/αα) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated α-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free α-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess α-globin, AHSP also acts as a molecular chaperone to stabilize nascent α-globin for HbA assembly. Our findings illustrate what we believe to be a novel adaptive mechanism by which a specialized cell coordinates high-level production of a multisubunit protein and protects against various synthetic imbalances.
PMCID: PMC1904324  PMID: 17607360

Results 1-10 (10)