Dataset Information

NMR solution structure, stability, and interaction of the recombinant bovine fibrinogen alphaC-domain fragment.

ABSTRACT: According to the existing hypothesis, in fibrinogen, the COOH-terminal portions of two Aalpha chains are folded into compact alphaC-domains that interact intramolecularly with each other and with the central region of the molecule; in fibrin, the alphaC-domains switch to an intermolecular interaction resulting in alphaC-polymers. In agreement, our recent NMR study identified within the bovine fibrinogen Aalpha374-538 alphaC-domain fragment an ordered compact structure including a beta-hairpin restricted at the base by a 423-453 disulfide linkage. To establish the complete structure of the alphaC-domain and to further test the hypothesis, we expressed a shorter alphaC-fragment, Aalpha406-483, and performed detailed analysis of its structure, stability, and interactions. NMR experiments on the Aalpha406-483 fragment identified a second loose beta-hairpin formed by residues 459-476, yielding a structure consisting of an intrinsically unstable mixed parallel/antiparallel beta-sheet. Size-exclusion chromatography and sedimentation velocity experiments revealed that the Aalpha406-483 fragment forms soluble oligomers whose fraction increases with an increase in concentration. This was confirmed by sedimentation equilibrium analysis, which also revealed that the addition of each monomer to an assembling alphaC-oligomer substantially increases its stabilizing free energy. In agreement, unfolding experiments monitored by CD established that oligomerization of Aalpha406-483 results in increased thermal stability. Altogether, these experiments establish the complete NMR solution structure of the Aalpha406-483 alphaC-domain fragment, provide direct evidence for the intra- and intermolecular interactions between the alphaC-domains, and confirm that these interactions are thermodynamically driven.

SUBMITTER: Burton RA

PROVIDER: S-EPMC2597398 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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NMR solution structure, stability, and interaction of the recombinant bovine fibrinogen alphaC-domain fragment.

Burton Robert A RA Tsurupa Galina G Hantgan Roy R RR Tjandra Nico N Medved Leonid L

Biochemistry 20070623 29

According to the existing hypothesis, in fibrinogen, the COOH-terminal portions of two Aalpha chains are folded into compact alphaC-domains that interact intramolecularly with each other and with the central region of the molecule; in fibrin, the alphaC-domains switch to an intermolecular interaction resulting in alphaC-polymers. In agreement, our recent NMR study identified within the bovine fibrinogen Aalpha374-538 alphaC-domain fragment an ordered compact structure including a beta-hairpin re ...[more]

PMID: 17590019

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Project description: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.

Project description:Many multidomain proteins and ribonucleic acids consist of domains that autonomously fold and that are linked together by flexible junctions. This architectural design allows domains to sample a wide range of positions with respect to one another, yet do so in a way that retains structural specificity, since the number of sampled conformations remains extremely small compared to the total conformations that would be sampled if the domains were connected by an infinitely long linker. This "tuned" flexibility in interdomain conformation is in turn used in many biochemical processes. There is great interest in characterizing the dynamic properties of multidomain systems, and moving beyond conventional descriptions in terms of static structures, toward the characterization of population-weighted ensembles describing a distribution of many conformations sampled in solution. There is also great interest in understanding the design principles and underlying physical and chemical interactions that specify the nature of interdomain flexibility. NMR spectroscopy is one of the most powerful techniques for characterizing motions in complex biomolecules and has contributed greatly toward our basic understanding of dynamics in proteins and nucleic acids and its role in folding, recognition, and signaling. Here, we review methods that have been developed in our laboratories to address these challenges. Our approaches are based on the ability of one domain of the molecule to self-align in a magnetic field, or to dominate the overall orientation of the molecule, so that the conformational freedom of other domains can be assessed by their degree of alignment induced by the aligned part. In turn, this self-alignment ability can be intrinsic or can be caused by tagging appropriate constructs to the molecule of interest. In general, self-alignment is due to magnetic susceptibility anisotropy. Nucleic acids with elongated helices have this feature, as well as several paramagnetic metal centers that can be found in, or attached to, a protein domain.

Dataset Information

NMR solution structure, stability, and interaction of the recombinant bovine fibrinogen alphaC-domain fragment.

Publications

NMR solution structure, stability, and interaction of the recombinant bovine fibrinogen alphaC-domain fragment.

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