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Characterization of the two mutants,...

Ratakonda, Sireesha.

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Characterization of the two mutants, Y29F and Y29A of Vitreoscilla hemoglobin.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Characterization of the two mutants, Y29F and Y29A of Vitreoscilla hemoglobin./
作者:	Ratakonda, Sireesha.
面頁冊數:	142 p.
附註:	Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: .
Contained By:	Dissertation Abstracts International72-01B.
標題:	Biology, General. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3435828
ISBN:	9781124344379

Characterization of the two mutants, Y29F and Y29A of Vitreoscilla hemoglobin.
Ratakonda, Sireesha.

Characterization of the two mutants, Y29F and Y29A of Vitreoscilla hemoglobin. - 142 p.

Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: .

Thesis (Ph.D.)--Illinois Institute of Technology, 2010.

Vitreoscilla, an obligately aerobic Gram-negative bacterium, synthesizes a relatively large amount of soluble, homodimeric hemoglobin (VHb) under low oxygen conditions. The ability of VHb to improve the growth properties of E. coli has important applications in biotechnology and increases the production of recombinant proteins and antibiotics. The three-dimensional structure of VHb, obtained through X-ray crystallography (1997), has shown that the structure conforms to the characteristic globin fold. The polypeptide segment connecting helices C and E, however is disordered and residues E7-E10 do not adopt the usual alpha helical conformation. A highly conserved residue in the VHb distal site, Tyr29 (B 10) may be involved in forming a hydrogen bond to the ligand. To study the importance of this residue (Tyr29 (B10)) in oxygen binding we have introduced two mutations at this site, namely Y29F and Y29A, to see if the side chain of Tyrosine (Tyr) has any role in stabilization of oxygen and if a structural change in this position can cause a major change in the functional properties of the protein. The high resolution crystal structures obtained from these two mutants show that the Y29F mutant crystallized isomorphously (P21) with the wild-type and had a dimer in the asymmetric unit while Y29A crystallized in a different spacegroup (C2) with a monomer in the asymmetric unit. All the missing residues in the disordered region (44-52) were built in and showed that the region is partly helical. Residues E7-E10, which are non-helical in the earlier reported wild-type VHb, are helical in this mutant. Also, E7 (Q53) is in close contact to the propionate on the heme group which is significantly different from the wild type and the Y29F mutant, where it faces toward the solvent. The Y29F mutant is, for the most part, identical in structure to wild-type VHb. Studies on these two mutants using CD and CO difference spectra have shown that there is not a major change in the helicity and the binding of heme in the two mutants, which emphasizes that some of the ordering seen in the Y29A mutant is an artifact of crystallization especially the stabilization of the M45 to the neighboring molecule. There is a compensatory rearrangement in the structure of Y29A mutant wherein the space occupied by the oxygen binding site in wild type (Tyr29 (B10)) is now occupied by Pro 54 in the mutant and the space occupied by Pro 54 is partly occupied by the Gln53.

ISBN: 9781124344379Subjects--Topical Terms:

1018625
Biology, General.

Characterization of the two mutants, Y29F and Y29A of Vitreoscilla hemoglobin.
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245 1 0 $a Characterization of the two mutants, Y29F and Y29A of Vitreoscilla hemoglobin.
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520 $a Vitreoscilla, an obligately aerobic Gram-negative bacterium, synthesizes a relatively large amount of soluble, homodimeric hemoglobin (VHb) under low oxygen conditions. The ability of VHb to improve the growth properties of E. coli has important applications in biotechnology and increases the production of recombinant proteins and antibiotics. The three-dimensional structure of VHb, obtained through X-ray crystallography (1997), has shown that the structure conforms to the characteristic globin fold. The polypeptide segment connecting helices C and E, however is disordered and residues E7-E10 do not adopt the usual alpha helical conformation. A highly conserved residue in the VHb distal site, Tyr29 (B 10) may be involved in forming a hydrogen bond to the ligand. To study the importance of this residue (Tyr29 (B10)) in oxygen binding we have introduced two mutations at this site, namely Y29F and Y29A, to see if the side chain of Tyrosine (Tyr) has any role in stabilization of oxygen and if a structural change in this position can cause a major change in the functional properties of the protein. The high resolution crystal structures obtained from these two mutants show that the Y29F mutant crystallized isomorphously (P21) with the wild-type and had a dimer in the asymmetric unit while Y29A crystallized in a different spacegroup (C2) with a monomer in the asymmetric unit. All the missing residues in the disordered region (44-52) were built in and showed that the region is partly helical. Residues E7-E10, which are non-helical in the earlier reported wild-type VHb, are helical in this mutant. Also, E7 (Q53) is in close contact to the propionate on the heme group which is significantly different from the wild type and the Y29F mutant, where it faces toward the solvent. The Y29F mutant is, for the most part, identical in structure to wild-type VHb. Studies on these two mutants using CD and CO difference spectra have shown that there is not a major change in the helicity and the binding of heme in the two mutants, which emphasizes that some of the ordering seen in the Y29A mutant is an artifact of crystallization especially the stabilization of the M45 to the neighboring molecule. There is a compensatory rearrangement in the structure of Y29A mutant wherein the space occupied by the oxygen binding site in wild type (Tyr29 (B10)) is now occupied by Pro 54 in the mutant and the space occupied by Pro 54 is partly occupied by the Gln53.
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