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1.  Tertiary and quaternary allostery in HbII from Scapharca inaequivalvis 
Biochemistry  2013;52(12):10.1021/bi301620x.
The clam Scapharca inaequivalvis possesses two cooperative oxygen binding hemoglobins in its red cells: a homodimeric HbI and a heterotetrameric A2B2 HbII. Each AB dimeric half of HbII is assembled very similarly to that of the well studied HbI. This study presents crystal structures of HbII along with oxygen binding data both in the crystalline state and in wet nanoporous silica gels. Despite very similar ligand-linked structural transitions observed in HbI and HbII crystals, HbII in the crystal or encapsulated in silica gels apparently exhibits minimal cooperativity in oxygen binding, in contrast with the full cooperativity exhibited by HbI crystals. However, oxygen binding curves in the crystal indicate the presence of a significant functional inequivalence of A and B chains. When this inequivalence is taken into account, both crystal and R state gel functional data are consistent with the conservation of a tertiary contribution to cooperative oxygen binding, quantitatively similar to that measured for HbI, and are in keeping with the structural information. Furthermore, our results indicate that to fully express the cooperative ligand binding, HbII requires quaternary transitions hampered by crystal lattice and gel encapsulation, revealing greater complexity in cooperative function than the direct communication across a dimeric interface observed in HbI.
PMCID: PMC3742685  PMID: 23458680
X-ray crystallography; microspectrophotometry; silica gel; cooperativity; hemoglobin
2.  Asymmetry of the Active Site Loop Conformation between Subunits of Glutamate-1-semialdehyde Aminomutase in Solution 
BioMed Research International  2013;2013:353270.
Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5′-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5′-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.
PMCID: PMC3747428  PMID: 23984351
3.  Design of O-acetylserine sulfhydrylase inhibitors by mimicking Nature 
Journal of medicinal chemistry  2010;53(1):345-356.
The inhibition of cysteine biosynthesis in prokaryotes and protozoa has been proposed to be relevant for the development of antibiotics. Haemophilus influenzae O-acetylserine sulfhydrylase (OASS), catalyzing L-cysteine formation, is inhibited by the insertion of the C-terminal pentapeptide (MNLNI) of serine acetyltransferase into the active site. 400 MNXXI pentapeptides were generated, docked into OASS active site using GOLD and scored with HINT. The terminal P5 Ile accounts for about 50% of the binding energy. Glu or Asp at position P4, and to a lesser extent, at position P3, also significantly contribute to the binding interaction. The predicted affinity of 14 selected pentapeptides correlated well with the experimentally determined dissociation constants. The X-ray structure of three high affinity pentapeptides-OASS complexes were compared with the docked poses. These results, combined with a GRID analysis of the active site, allowed us to define a pharmacophoric scaffold for the design of peptidomimetic inhibitors.
PMCID: PMC2804909  PMID: 19928859
4.  Haemoglobin-based oxygen carriers: research and reality towards an alternative to blood transfusions 
Blood Transfusion  2010;8(Suppl 3):s59-s68.
PMCID: PMC2897202  PMID: 20606751
haemoglobin; oxygen therapy; transfusion; red blood cells

Results 1-4 (4)