Heme binding by the N-terminal fragment 1-44 of human growth hormone.
Fragment 1-44 of human growth hormone (hGH), prepared in vitro by limited proteolysis of the hormone with pepsin at low pH, encompasses in full the N-terminal helix of this four-helix bundle protein [Spolaore, B., Polverino de Laureto, P., Zambonin, M., and Fontana, A. (2004) Biochemistry 40, 9460-9468]. Here, we report the new and interesting observation that fragment 1-44 can bind heme. The binding property is specific for the N-terminal helix of hGH, since heme binding does not occur with fragment 45-191 or the entire protein. The spectral characteristics of Fe-protoporphyrin IX are those of a low-spin, hexacoordinated iron ligated by two imidazole rings of His residues or His and Met residues. Far-UV circular dichroism (CD) measurements revealed that fragment 1-44 acquires a helical secondary structure upon heme binding. Heme appears to be bound to the fragment in a stereospecific way, since an induced dichroic signal is observed in the Soret region of the CD spectrum. The heme-fragment complex occurs in a 1:1 molar ratio, as determined by spectrophotometric titration, as well as by electrospray-ionization mass spectrometric analysis of the complex. The fragment alone is much more susceptible to tryptic digestion than the heme complex, implying a more folded and rigid structure of this last species. It is proposed that the molecular features of fragment 1-44 determining its heme-binding property reside in the amphipathic character of the helix adopted by the fragment, as well as in the presence in its polypeptide chain of His18, His21, and Met14. These residues can act as specific ligands for the heme-iron, as observed with cytochromes.
Spolaore
Barbara
B
CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy.
De Filippis
Vincenzo
V
Fontana
Angelo
A
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006418:Heme / Q000378:metabolism*; D019382:Human Growth Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D006418:Heme