1XCH

MYOGLOBIN (HORSE HEART) MUTANT WITH LEU 104 REPLACED BY ASN (L104N)

1XCH の概要

• エントリーDOI: 10.2210/pdb1xch/pdb
• 分子名称: MYOGLOBIN, SULFATE ION, PROTOPORPHYRIN IX CONTAINING FE, ... (4 entities in total)
• 機能のキーワード: heme, oxygen transport, respiratory protein, muscle
• 由来する生物種: Equus caballus (horse)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 17697.01

構造登録者

Maurus, R.,Brayer, G.D. (登録日: 1997-06-06, 公開日: 1997-09-17, 最終更新日: 2024-02-14)

主引用文献

Maurus, R.,Overall, C.M.,Bogumil, R.,Luo, Y.,Mauk, A.G.,Smith, M.,Brayer, G.D.
A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket.
Biochim.Biophys.Acta, 1341:1-13, 1997

PubMed Abstract: Well-ordered internal amino acids can contribute significantly to the stability of proteins. To investigate the importance of the hydrophobic packing interface between helices G and H in the proximal heme pocket of horse heart myoglobin, the highly conserved amino acid, Leu104, was substituted with asparagine, a polar amino acid of similar size. The Leu104Asn mutant protein and its recombinant wild-type horse heart myoglobin counterpart were expressed from synthetic genes in Escherichia coli. Thermal denaturation of these two recombinant myoglobins, as studied by measurement of circular dichroism ellipticity at 222 nm, revealed that the Leu104Asn mutant had a significantly lower t(m) (71.8 +/- 1 degree C, pH 7.0) than recombinant wild-type myoglobin (81.3 +/- 1 degree C, pH 7.0). To examine the extent to which this 10 degrees C decrease in thermal stability was associated with structural perturbations, X-ray diffraction techniques were used to determine the three-dimensional structures of both the recombinant wild-type and Leu104Asn myoglobins to 0.17 nm resolution. Refinement of these structures gave final crystallographic R-factors of 16.0% and 17.9%, respectively. Structural comparison of the natural and recombinant wild-type myoglobins, together with absorption spectroscopic and electron paramagnetic resonance (EPR) analyses, confirmed the proper expression and folding of the recombinant protein in E. coli. Surprisingly, despite the decreased thermal stability of the Leu104Asn mutant, there are no significant structural differences between the mutant and wild-type myoglobins. EPR and absorption spectroscopic analyses further confirmed the similar nature of the heme iron centres in both proteins. Thus, the introduction of an energetically unfavourable change in side chain polarity at position 104 into a hydrophobic environment that does not support the hydrogen bonding potential of the mutant asparagine appears to perturb important stabilizing helix-helix and heme-protein interactions. The induced structural destabilization is thereby reflected by a significant decrease in the t(m) of horse heart myoglobin.

PubMed: 9300804
DOI: 10.1016/S0167-4838(97)00064-2

実験手法

X-RAY DIFFRACTION (1.7 Å)