title: Structural insights into fibrinogen dynamics using amide hydrogen/deuterium exchange mass spectrometry.
abstract: We determined the amide hydrogen/deuterium exchange profile of native human fibrinogen under physiologic conditions. After optimization of the quench and proteolysis conditions, more than 1,200 peptides were identified by mass spectrometry, spanning more than 90% of the constituent Aα, Bβ, and γ chain amino acid sequences. The compact central and distal globular regions of fibrinogen were well protected from deuterium exchange, with the exception of the unfolded amino-terminal segments of the Aα and Bβ chains extending from the central region, and the short γ chain "tail" extending from each distal globular region. The triple-helical coiled-coil regions, which bridge the central region to each distal region, were also well protected with the exception of a moderately fast-exchanging area in the middle of each coiled-coil adjacent to the γ chain carbohydrate attachment site. These dynamic regions appear to provide flexibility to the fibrinogen molecule. The γ chain "out loop" contained within each coiled-coil also exchanged rapidly. The αC domain (Aα 392-610) exchanged rapidly, with the exception of a short segment sandwiched between a conserved disulfide linkage in the N-terminal αC subdomain. This latter finding is consistent with a mostly disordered structure for the αC domain in native fibrinogen. Analysis of the dysfibrinogen Bβ 235 Pro/Leu, which exhibits abnormal fibrin structure, revealed enhanced deuterium exchange surrounding the Pro/Leu substitution site as well as in the vicinity of the high affinity calcium binding site and the A knob polymerization pocket within the γC domain. The implication of these changes with respect to fibrin structure is discussed.
authors.affiliation: Department of Medicine, University of California at San Diego, La Jolla, CA 92093, USA. jjmarsh@ucsd.edu
authors.forename: James J
authors.initials: JJ
authors.lastname: Marsh
authors.forename: Henry S
authors.initials: HS
authors.lastname: Guan
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: Peter G
authors.initials: PG
authors.lastname: Chiles
authors.forename: Danny
authors.initials: D
authors.lastname: Tran
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Morris
mesh_terms: D001665:Binding Sites; D041961:Deuterium Exchange Measurement; D005337:Fibrin / Q000737:chemistry / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000235:genetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D010446:Peptide Fragments; D005337:Fibrin; D005340:Fibrinogen; D011392:Proline; D007930:Leucine