title: The two tryptophans of β2-microglobulin have distinct roles in function and folding and might represent two independent responses to evolutionary pressure.
abstract: BACKGROUND
  We have recently discovered that the two tryptophans of human β2-microglobulin have distinctive roles within the structure and function of the protein. Deeply buried in the core, Trp95 is essential for folding stability, whereas Trp60, which is solvent-exposed, plays a crucial role in promoting the binding of β2-microglobulin to the heavy chain of the class I major histocompatibility complex (MHCI). We have previously shown that the thermodynamic disadvantage of having Trp60 exposed on the surface is counter-balanced by the perfect fit between it and a cavity within the MHCI heavy chain that contributes significantly to the functional stabilization of the MHCI. Therefore, based on the peculiar differences of the two tryptophans, we have analysed the evolution of β2-microglobulin with respect to these residues.
  
  
  RESULTS
  Having defined the β2-microglobulin protein family, we performed multiple sequence alignments and analysed the residue conservation in homologous proteins to generate a phylogenetic tree. Our results indicate that Trp60 is highly conserved, whereas some species have a Leu in position 95; the replacement of Trp95 with Leu destabilizes β2-microglobulin by 1 kcal/mol and accelerates the kinetics of unfolding. Both thermodynamic and kinetic data fit with the crystallographic structure of the Trp95Leu variant, which shows how the hydrophobic cavity of the wild-type protein is completely occupied by Trp95, but is only half filled by Leu95.
  
  
  CONCLUSIONS
  We have established that the functional Trp60 has been present within the sequence of β2-microglobulin since the evolutionary appearance of proteins responsible for acquired immunity, whereas the structural Trp95 was selected and stabilized, most likely, for its capacity to fully occupy an internal cavity of the protein thereby creating a better stabilization of its folded state.
authors.affiliation: Department of Biochemistry, University of Pavia, via Taramelli 3b, 27100 Pavia, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Raimondi
authors.forename: Nicola
authors.initials: N
authors.lastname: Barbarini
authors.forename: Palma
authors.initials: P
authors.lastname: Mangione
authors.forename: Gennaro
authors.initials: G
authors.lastname: Esposito
authors.forename: Stefano
authors.initials: S
authors.lastname: Ricagno
authors.forename: Martino
authors.initials: M
authors.lastname: Bolognesi
authors.forename: Irene
authors.initials: I
authors.lastname: Zorzoli
authors.forename: Loredana
authors.initials: L
authors.lastname: Marchese
authors.forename: Cristina
authors.initials: C
authors.lastname: Soria
authors.forename: Riccardo
authors.initials: R
authors.lastname: Bellazzi
authors.forename: Maria
authors.initials: M
authors.lastname: Monti
authors.forename: Monica
authors.initials: M
authors.lastname: Stoppini
authors.forename: Mario
authors.initials: M
authors.lastname: Stefanelli
authors.forename: Paolo
authors.initials: P
authors.lastname: Magni
authors.forename: Vittorio
authors.initials: V
authors.lastname: Bellotti
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000378:metabolism; D000818:Animals; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011487:Protein Conformation; D017510:Protein Folding; D016415:Sequence Alignment; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000682:Amyloid; D001613:beta 2-Microglobulin; D014364:Tryptophan