title: Structure, stability, and interaction of the fibrin(ogen) alphaC-domains.
abstract: Our recent study established the NMR structure of the recombinant bAalpha406-483 fragment corresponding to the NH(2)-terminal half of the bovine fibrinogen alphaC-domain and revealed that at increasing concentrations this fragment forms oligomers (self-associates). The major goals of the study presented here were to determine the structure and self-association of the full-length human fibrinogen alphaC-domains. To accomplish these goals, we prepared a recombinant human fragment, hAalpha425-503, homologous to bovine bAalpha406-483, and demonstrated using NMR, CD, and size-exclusion chromatography that its overall fold and ability to form oligomers are similar to those of bAalpha406-483. We also prepared recombinant hAalpha392-610 and bAalpha374-568 fragments corresponding to the full-length human and bovine alphaC-domains, respectively, and tested their structure, stability, and ability to self-associate. Size-exclusion chromatography revealed that both fragments form reversible oligomers in a concentration-dependent manner. Their oligomerization was confirmed in sedimentation equilibrium experiments, which also established the self-association affinities of these fragments and revealed that the addition of each monomer to assembling alphaC-oligomers substantially increases the stabilizing free energy. In agreement, unfolding experiments monitored by CD established that self-association of both fragments results in a significant increase in their thermal stability. Analysis of CD spectra of both fragments revealed that alphaC self-association results in an increase in the level of regular structure, implying that the COOH-terminal half of the alphaC-domain adopts an ordered conformation in alphaC-oligomers and that this domain contains two independently folded subdomains. Altogether, these data further clarify the structure of the human and bovine alphaC-domains and the molecular mechanism of their self-association into alphaC-polymers in fibrin.
authors.affiliation: Center for Vascular and Inflammatory Diseases and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 800 West Baltimore Street, Baltimore, Maryland 21201, USA.
authors.forename: Galina
authors.initials: G
authors.lastname: Tsurupa
authors.forename: Roy R
authors.initials: RR
authors.lastname: Hantgan
authors.forename: Robert A
authors.initials: RA
authors.lastname: Burton
authors.forename: Igor
authors.initials: I
authors.lastname: Pechik
authors.forename: Nico
authors.initials: N
authors.lastname: Tjandra
authors.forename: Leonid
authors.initials: L
authors.lastname: Medved
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary
chemical_list: D005340:Fibrinogen