Unusual stability of human neuroglobin at low pH--molecular mechanisms and biological significance.
Neuroglobin (Ngb) is a recently discovered globin that is predominantly expressed in the brain, retina and other nerve tissues of human and other vertebrates. Ngb has been shown to act as a neuroprotective factor, promoting neuronal survival in conditions of hypoxic-ischemic insult, such as those occurring during stroke. In this work, the conformational and functional stability of Ngb at acidic pH was analyzed, and the results were compared to those obtained with Mb. It was shown by spectroscopic and biochemical (limited proteolysis) techniques that, at pH 2.0, apoNgb is a folded and rigid protein, retaining most of the structural features that the protein displays at neutral pH. Conversely, apoMb, under the same experimental conditions of acidic pH, is essentially a random coil polypeptide. Urea-mediated denaturation studies revealed that the stability displayed by apoNgb at pH 2.0 is very similar to that of Mb at pH 7.0. Ngb also shows enhanced functional stability as compared with Mb, being capable of heme binding over a more acidic pH range than Mb. Furthermore, Ngb reversibly binds oxygen at acidic pH, with an affinity that increases as the pH is decreased. It is proposed that the acid-stable fold of Ngb depends on the particular amino acid composition of the protein polypeptide chain. The functional stability at low pH displayed by Ngb was instead shown to be related to hexacoordination of the heme group. The biological implications of the unusual acid resistance of the folding and function of Ngb are discussed.
CRIBI Biotechnology Center, University of Padua, Italy.
Paola
P
Picotti
Sylvia
S
Dewilde
Angela
A
Fago
Christian
C
Hundahl
Vincenzo
V
De Filippis
Luc
L
Moens
Angelo
A
Fontana
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005914:Globins / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D000078787:Neuroglobin; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D014508:Urea / Q000378:metabolism
D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D006418:Heme; D014508:Urea; D005914:Globins; D010100:Oxygen