title: Structural characterization and unfolding mechanism of human 4F2hc ectodomain.
abstract: 4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, β1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% β-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57-59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M(-1)). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (βα)(8) barrel; C, antiparallel β(8) sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the β-barrel of domain A followed by additional hydrophobic interactions in domain C.
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense, 28040-Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Turnay
authors.forename: Joana
authors.initials: J
authors.lastname: Fort
authors.forename: Nieves
authors.initials: N
authors.lastname: Olmo
authors.forename: Angélica
authors.initials: A
authors.lastname: Santiago-Gómez
authors.forename: Manuel
authors.initials: M
authors.lastname: Palacín
authors.forename: M A Antonia
authors.initials: MA
authors.lastname: Lizarbe
mesh_terms: D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D027282:Fusion Regulatory Protein 1, Heavy Chain; D011994:Recombinant Proteins; C545989:SLC3A2 protein, human