Parakeet Hemoglobin - Its Crystal Structure and Oxygen Affinity in Relation to Some Avian Hemoglobins.

BACKGROUND: "Avians" often show efficient oxygen management to meet the demands of their metabolism. Hemoglobin, a transporter protein consists of four non-covalently linked subunits contain haem binding hydrophobic pocket serves as a site of allosteric cooperativity. The physiology and anatomy of both mammals and avian are functionally different, in birds, the respiratory system formed by small air sacs that serve as tidal ventilation for the lungs and have no significant exchange across their cells. Parakeet (Psittacula krameri) a tropical and non-migrating species and it is easily adapted to living in disturbed habitat. The sequence analysis reveals that α and ß chain of parakeet hemoglobin highly similar grey lag goose and bar headed goose hemoglobin respectively. Thus it has been tempted us to study in to analyzing the sequence and structural comparison of this hemoglobin to find out the physiological capabilities of parakeet hemoglobin. OBJECTIVE: The structure determination studies of parakeet hemoglobin by X-ray diffraction. The sequence and structure are compared with goose, chicken and human Hb, emphasizing the role of amino acids in the subunit contacts that facilitate survival by low oxygen demand. METHODS: The Hb was purified and crystallized by hanging drop vapor diffusion method using poly ethylene glycol (PEG) 3350 and sodium phosphate buffer. X-ray diffracted data set was collected at 3Å resolution, the data was processed in Automar and molecular replacement, refinements, model building was carried out in CCP4i program package. The final refined model was deposited in protein data bank with accession id 2zfb. RESULTS: The tertiary structure of Parakeet Hb is compared with the met form of BHG Hb (1c40) and oxy form of GLG (1faw) and oxy form of human Hbs (1hho). Superimposing parakeet Hb α1ß1 subunit with 'R' state human Hb shows an r.m.s.d of 0.98 Å and for BHG and GLG Hb, the r.m.s.d shows 0.72 and 0.61 Å. The replacement of α115Asp in parakeet Hb as against the α115Glu in human Hb results in the movement of GH corners. The amino acid proline at α50 present only in Parakeet Hb and Chicken HbD and not present in any other avian family which includes human Hb. The residue α78Thr located in EF corner loop region, which slightly diverge when superimposing with human and BHG Hb and also replacement of α113Asn present only in Parakeet Hb placed near the FG helix corner. CONCLUSION: The present study describes the structure determination of parakeet hemoglobin and its structural features to understand its oxygen affinity characteristics. The crystals were obtained by buffered low-salt conditions, like those of chicken HbD, carbonmonoxy and cyanomet human Hb. The present study reveals several interesting and unique modifications in the finer aspects of the quaternary structure of parakeet Hb, which are involved in oxygen affinity characteristics and the α1ß1 subunit contacts. Crystallization of parakeet Hb with allosteric effectors like Inositol pentaphosphate may bring further understanding of the influence of physiological and environmental factors on the quaternary structure.

Insights of structure-based pharmacophore studies and inhibitor design against Gal3 receptor through molecular dynamics simulations.

Our present work studies the structure-based pharmacophore modeling and designing inhibitor against Gal3 receptor through molecular dynamics (MD) simulations extensively. Pharmacophore models play a key role in computer-aided drug discovery like in the case of virtual screening of chemical databases, de novo drug design and lead optimization. Structure-based methods for developing pharmacophore models are important, and there have been a number of studies combining such methods with the use of MD simulations to model protein's flexibility. The two potential antagonists SNAP 37889 and SNAP 398299 were docked and simulated for 250 ns and the results are analyzed and carried for the structure-based pharmacophore studies. This helped in identification of the subtype selectivity of the binding sites of the Gal3 receptor. Our work mainly focuses on identifying these binding site residues and to design more potent inhibitors compared to the previously available inhibitors through pharmacophore models. The study provides crucial insight into the binding site residues Ala2, Asp3, Ala4, Gln5, Phe24, Gln79, Ala80, Ile82, Tyr83, Trp88, His99, Ile102, Tyr103, Met106, Tyr157, Tyr161, Pro174, Trp176, Arg181, Ala183, Leu184, Asp185, Thr188, Trp248, His251, His252, Ile255, Leu256, Phe258, Trp259, Tyr270, Arg273, Leu274 and His277, which plays a significant role in the conformational changes of the receptor and helps to understand the inhibition mechanism. Communicated by Ramaswamy H. Sarma.

Understanding the dual mechanism of bioactive peptides targeting the enzymes involved in Renin Angiotensin System (RAS): An in-silico approach.

Understanding the dual inhibition mechanism of food derivative peptides targeting the enzymes (Renin and Angiotensin Converting enzyme) in the Renin Angiotensin System. Two peptides RALP and WYT were reported to possess antihypertensive activity targeting both renin and ACE, and we have used molecular docking and molecular dynamics simulation, in order to understand the underlying mechanism. The selected peptides (RALP and WYT) from the series of peptides reported were docked to renin and ACE and two binding modes were selected based on the binding energy, interaction pattern and clusters of docking simulation. The enzyme-peptide complexes for renin and ACE (Renin/RALP1,2; ACE/RALP1,2; Renin/WYT1,2 and ACE/WYT1,2) were subjected to molecular dynamics simulation. Our results identified that the peptides inhibiting renin, tends to move out of the binding pockets (S1' S2') which is critical for potent binding and occupies the less important pockets (S4 and S3). This could possibly be the reason for its low potency. Whereas, the same peptides targeting ACE, tends to be intact in the pocket because of the metal ion coordination and there is an ample room to improve on its efficacy. Our results further pave way for the biochemist, medicinal chemist to design dual peptides targeting the RAS effectively. Communicated by Ramaswamy H. Sarma.

Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of catfish (Clarias magur) haemoglobin.

Haemoglobin is an interesting physiologically significant protein composed of specific functional prosthetic haem and globin moieties. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of fish haemoglobins (Hbs). Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on Clarias magur Hb. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 2000 and NaCl as precipitants. The crystals belonged to the primitive monoclinic system P2, with unit-cell parameters a=98.35, b=56.63, c=112.88 Å, ß=100.22°; a complete data set was collected to a resolution of 2.4 Å. The Matthews coefficient of 2.42 Å3 Da(-1) for the crystal indicated the presence of two α2ß2 tetramers in the asymmetric unit.

Aggregation and self assembly of non-enzymatic glycation of collagen in the presence of amino guanidine and aspirin: an in vitro study.

Non-enzymatic glycation of collagen has been used in modern biomaterials science. This paper deals with in vitro studies on the effects of amino guanidine (AG) and aspirin in the non-enzymatic glycation (NEG) of collagen using thermal, conformational, fluorescence, turbidity and powder XRD measurements. There is no significant change in the fluorescence emission spectra for different concentrations of AG treated NEG of collagen whereas the emission intensity decreases as the concentration of aspirin increases. Circular dichroism (CD) revealed the disappearance of the positive peak at 220nm for glycated collagen in the presence of amino guanidine and aspirin suggesting the collapse of triple helical configuration. Nearly 15 degrees C decrease is observed in shrinkage temperature of glycated rat tail tendon (RTT) collagen fibres in the presence of aspirin. Powder XRD of glycated collagen nano-fibrils in the presence of amino guanidine reveals high crystalline nature and the enhancement of self assembly processes when compared to aspirin. To the best of our knowledge, this is the first report of powder XRD of the self assembly of collagen nano-fibrils without mineralization. Our experimental results suggest that in the non-enzymatic glycation of collagen both AG and aspirin play a pivotal role in the aggregation and self assembly processes. From the present study, it is possible to conclude that while AG significantly influences the self assembly processes, aspirin facilitates the aggregation processes.

Purification, crystallization and preliminary X-ray diffraction studies of parakeet (Psittacula krameri) haemoglobin.

Birds often show efficient oxygen management in order to meet the special demands of their metabolism. However, the structural studies of avian haemoglobins (Hbs) are inadequate for complete understanding of the mechanism involved. Towards this end, purification, crystallization and preliminary X-ray diffraction studies have been carried out for parakeet Hb. Parakeet Hb was crystallized as the met form in low-salt buffered conditions after extracting haemoglobin from crude blood by microcentrifugation and purifying the sample by column chromatography. Good-quality crystals were grown from 10% PEG 3350 and a crystal diffracted to about 2.8 A resolution. Preliminary diffraction data showed that the Hb crystal belonged to the monoclinic system (space group C2), with unit-cell parameters a = 110.68, b = 64.27, c = 56.40 A, beta = 109.35 degrees . Matthews volume analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.