Chemical synthesis of mouse cripto CFC variants.
We report for the first time the chemical synthesis of refolded CFC domain of mouse Cripto (mCFC) and of two variants bearing mutations on residues W107 and H104 involved in Alk4 binding. The domains undergo spontaneous and quantitative refolding in about 4 h, yet with very different kinetics. Disulfide linkages have been assessed by enzyme digestion and mass spectrometry analysis of resulting fragments, and the first experimental studies on structural organization have been conducted by circular dichroism spectroscopy under different pH conditions. Upon refolding, the domains considerably change their conformations, although they do not assume canonical structures, and become highly resistant to enzyme degradation. A comparative study of receptor binding shows that the CFC domain can bind Alk4 and confirms the importance of W107 and H104 for receptor recognition.
Marasco
Daniela
D
Istituto di Biostrutture e Bioimmagini del CNR, Sezione Biostrutture, Napoli, Italy.
Saporito
Angela
A
Ponticelli
Salvatore
S
Chambery
Angela
A
De Falco
Sandro
S
Pedone
Carlo
C
Minchiotti
Gabriella
G
Ruvo
Menotti
M
D030201:Activin Receptors, Type I / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D010455:Peptides; D011994:Recombinant Proteins; C489450:Tdgf1 protein, mouse; D004815:Epidermal Growth Factor; D030201:Activin Receptors, Type I; C498933:Acvr1b protein, mouse