Structural organization of the fibrin(ogen) alpha C-domain.
We hypothesized that the alpha C-domain of human fibrinogen (residues hA alpha 221-610) and of other species consists of a compact COOH-terminal region (hA alpha 392-610) and a flexible NH(2)-terminal connector region (hA alpha 221-391) which may contain some regular structure [Weisel and Medved (2001) Ann. N.Y. Acad. Sci. 936, 312-327]. To test this hypothesis, we expressed in E. coli recombinant fragments corresponding to the full-length human alpha C-domain and its NH(2)- and COOH-terminal regions as well as their bovine counterparts, bA alpha 224-568, bA alpha 224-373, and bA alpha 374-568(538), respectively, and tested their folding status by fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC). All three methods revealed heat-induced unfolding transitions in the full-length bA alpha 224-568 and its two COOH-terminal fragments, indicating that the COOH-terminal portion of the bovine alpha C-domain is folded into a compact cooperative structure. Similar results were obtained by CD and DSC with the full-length and the COOH-terminal h392-610 human fragments. The NH(2)-terminal fragments of both species, b224-373 and h221-392, did not exhibit any sign of a compact structure. However, their heat capacity functions, CD spectra, and temperature dependence of ellipticity at 222 nm were typical for peptides in the extended helical poly(L-proline) type II conformation (PPII), suggesting that they contain this type of regular structure. This is consistent with the presence of proline-rich tandem repeats in the sequence of both bovine and human connector regions. These results indicate that both bovine and human fibrinogen alpha C-domains consist of a compact globular cooperative unit attached to the bulk of the molecule by an extended NH(2)-terminal connector region with a PPII conformation.
Tsurupa
Galina
G
Department of Biochemistry, The American Red Cross Holland Laboratory, Rockville, Maryland 20855, USA.
Tsonev
Latchezar
L
Medved
Leonid
L
D000595:Amino Acid Sequence; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002942:Circular Dichroism; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D010446:Peptide Fragments; D011994:Recombinant Proteins; C098000:fibrinogen alphaC; D005340:Fibrinogen