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Primary hyperparathyroidism ( PHPT) is a common endocrine neoplastic disorder caused by a failure of calcium sensing secondary to tumour development in one or more of the parathyroid glands. Parathyroid adenomas are comprised of distinct cellular subpopulations of variable clonal status that exhibit differing degrees of calcium responsiveness. To gain a clearer understanding of the relationship among cellular identity, tumour composition and clinical biochemistry in PHPT, we developed a novel single cell platform for quantitative evaluation of calcium sensing behaviour in freshly resected human parathyroid tumour cells. Live-cell intracellular calcium flux was visualized through Fluo-4- AM epifluorescence, followed by in situ immunofluorescence detection of the calcium sensing receptor ( CASR), a central component in the extracellular calcium signalling pathway. The reactivity of individual parathyroid tumour cells to extracellular calcium stimulus was highly variable, with discrete kinetic response patterns observed both between and among parathyroid tumour samples. CASR abundance was not an obligate determinant of calcium responsiveness. Calcium EC50 values from a series of parathyroid adenomas revealed that the tumours segregated into two distinct categories. One group manifested a mean EC50 of 2.40 mM (95% CI: 2.37-2.41), closely aligned to the established normal range. The second group was less responsive to calcium stimulus, with a mean EC50 of 3.61 mM (95% CI: 3.45-3.95). This binary distribution indicates the existence of a previously unappreciated biochemical sub-classification of PHPT tumours, possibly reflecting distinct etiological mechanisms. Recognition of quantitative differences in calcium sensing could have important implications for the clinical management of PHPT. [ABSTRACT FROM AUTHOR]

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ∼12 nts of RNA, shorter than the 24–28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions. [ABSTRACT FROM AUTHOR]

Alpha hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds free α hemoglobin (αHb) to maintain its structure and limit its pro-oxidant activity. Prior studies have defined two different αHb·AHSP complexes. Binding of AHSP to Fe(II) αHb induces an unusual configuration in which the F helix of the globin becomes disordered and the heme ring becomes solvent-exposed. Over time, this intermediate oxidizes to form a stable hemichrome in which the proximal (F8) and distal (E7) histidines are coordinated to the heme iron atom. The addition of βHb to either Fe(II) or Fe(III) αHb·AHSP displaces AHSP to generate tetrameric (α2β2) HbA species. The biochemical properties and in vivo significance of the two αHb·AHSP complexes are poorly understood. Here we show that Fe(III) αHb·AHSP forms from auto-oxidation of oxygenated αHb bound to AHSP and that this process is greatly accelerated at physiologic temperature and oxygen pressures. In contrast to free Fe(III) αHb hemichromes, AHSP-bound Fe(III) αHb does not precipitate and can be recycled into functional HbA. This requires enzymatic reduction of AHSP-bound αHb, either prior to or after extraction by β subunits. In contrast, reaction of Fe(II) αHb-AHSP with βHb generates functional HbA directly. Our findings support a model in which AHSP can either stabilize αHb transiently en route to HbA formation during normal erythropoiesis or convert excessive free αHb into a more chemically inert state from which recovery of αHb is possible by redox cycling. [ABSTRACT FROM AUTHOR]

The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined system- atically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O2, CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20-500-fold increases in the rates of O2 dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O2 in both the a and β chains (ΔGHis(E7)H-bond ≈-8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in k'O2, kNO, and calculated bimolecular rates of CO entry (k'entry) are observed. Replacing His(E7) with Trp causes further decreases in k'O2, k'NO, and k'entry to 1-2 μM-1 s-1 in β subunits, whereas ligand rebinding to αTrp(E7) subunits after photolysis is markedly biphasic, with fast k'O2, kCO, and k'NO values ≈150 μM-1 s-1 and slow rate constants ≈0.1 to 1 μM-1 s-1. Rapid bimolecular rebinding to an open α subunit conformation occurs immediately after photolysis of the αTrp(E7) mutant at high ligand concentrations. However, at equilibrium the closed αTrp(E7) side chain inhibits the rate of ligand binding >200-fold. These data suggest strongly that the E7 side chain functions as a gate for ligand entry in both HbA subunits. [ABSTRACT FROM AUTHOR]

Presents a study which examines the unfolding behavior of 10 different distal heme pocket mutants of sperm whale deoxymyoglobin (deoxyMb). Reference to the effects of distal histidine (His 64) replacement being the primary focus; Tracking of the formation of the spectroscopically distinguishable heme intermediate (I') of deoxyMb; Suggestion of observations.

The iron-regulated surface proteins IsdA, IsdB, and IsdC and transporter IsdDEF of Staphylococcus aureus are involved in heme acquisition. To establish an experimental model of heme acquisition by this system, we have investigated hemin transfer between the various couples of human methemoglobin (metHb), IsdA, IsdB, IsdC, and IsdE by spectroscopic and kinetic analyses. The efficiencies of hemin transfer from hemin-contaming donors (holo-protein) to different hemin-free acceptors (apo-protein) were examined, and the rates of the transfer reactions were compared with that of indirect loss of hemin from the relevant donor to H64Y/V68F apomyoglobin. The efficiencies, spectral changes, and kinetics of the transfer reactions demonstrate that: 1) metHb directly transfers hemin to apo-IsdB, but not to apo-IsdA, apo-IsdC, and apo-IsdE; 2) holo-IsdB directly transfers hemin to apo-IsdA and apo-IsdC, but not to apo-IsdE; 3) apo-IsdE directly acquires hemin from holo-IsdC, but not from holo-IsdB and holo-IsdA; and 4) IsdB and IsdC enhance hemin transfer from metHb to apo-IsdC and from holo-IsdB to apo-IsdE, respectively. Taken together with our recent finding that holo-IsdA directly transfers its hemin to apo-IsdC, these results provide direct experimental evidence for a model in which IsdB acquires hemin from metHb and transfers it directly or through IsdA to IsdC. Hemin is then relayed to IsdE, the lipoprotein component of the IsdDEF transporter. [ABSTRACT FROM AUTHOR]

The surface protein Shp of Streptococcus pyogenes rapidly transfers its hemin to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. The structural basis and molecular mechanism of this hemin transfer have been explored by mutagenesis and truncation of Shp. The heme-binding domain of Shp is in the amino-terminal region and is functionally active by itself, although inclusion of the COOH-terminal domain speeds up the process 10-fold. Single alanine replacements of the axial methionine 66 and 153 ligands (ShpM66A and ShpM153A) cause formation of pentacoordinate hemin-Met complexes. The association equilibrium constants for hemin binding to wild-type, M66A, and M153A Shp are 5,300, 22,000, and 38 μM-1, respectively, showing that the Met153-Fe bond is critical for high affinity binding and that Met66 destabilizes hemin binding to facilitate its rapid transfer. ShpM66A and ShpM153A rapidly bind to hemin-free HtsA (apoHtsA), forming stable transfer intermediates. These intermediates appear to be Shp-hemin- HtsA complexes with one axial ligand from each protein and decay to the products with rate constants of 0.47–3 s-1. Thus, the M66A and M153A replacements alter the kinetic mechanism and unexpectedly slow down hemin transfer by stabilizing the intermediates. These results, in combination with the structure of the Shp heme-binding domain, allow us to propose a ‘plug-in’ mechanism in which side chains from apoHtsA are inserted into the axial positions of hemin in Shp to extract it from the surface protein and pull it into the transporter active site. [ABSTRACT FROM AUTHOR]

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 ↵ 65 °C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B ↵ A, C ↵ A, and D ↵ A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B ↵ A and C ↵ A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly. [ABSTRACT FROM AUTHOR]

The hemoglobin family of proteins, ubiquitous in all domains of life, evolved from an ancestral protein of primordial function to extant hemoglobins that perform a myriad of functions with diverged biochemical properties. Study of homologs in bacterial hyperthermophiles may shed light on both mechanisms of adaptation to extreme conditions and the nature of the ancestral protein. A hemoglobin was identified in Aquifex aeolicus, cloned, recombinantly expressed, purified, and characterized. This hemoglobin is monomeric, resistant to thermal and chemical denaturation, pentacoordinate in the ferrous deoxygenated state, and oxygen-avid. The oxygen equilibrium dissociation constant is approximately 1 nM at room temperature, due in part to a hydrogen bond between the bound ligand and a tyrosine residue in the distal pocket. These biochemical properties of A. aeolicus thermoglobin, AaTgb, may have been shared by the ancestral hemoglobin, thus suggesting possible primordial functions and providing a starting point for consequent evolution of the hemoglobin family. [ABSTRACT FROM AUTHOR]

The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O2 dissociation and ultra-high O2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O2 dissociation (kO2 ≈ 200–600 s-1) and moderate O2 affinity (KO2 ≈1 μM-1) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, kO2 decreases 1000-fold and KO2 increases 130-fold at pH 7.0, 20 °C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 → Val CerHbO2 at 1.70 Å resolution is almost identical to that of the wild-type protein (root mean square deviation of 0.12 Å). The dramatic functional and spectral effects of the Thr-E11 → Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val ‘frees’ the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O2 affinity, a decrease of the rate coefficient for O2 dissociation, a 40 cm-1 decrease in Vco of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites. [ABSTRACT FROM AUTHOR]