Folding of epidermal growth factor-like repeats from human tenascin studied through a sequence frame-shift approach.
In order to investigate the factors that determine the correct folding of epidermal growth factor-like (EGF) repeats within a multidomain protein, we prepared a series of six peptides that, taken together, span the sequence of two EGF repeats of human tenascin, a large protein from the extracellular matrix. The peptides were selected by sliding a window of the average length of tenascin EGF repeats over the sequence of EGF repeats 13 and 14. We thus obtained six peptides, EGF-f1 to EGF-f6, that are 33 residues long, contain six cysteines each, and bear a partial overlap in the sequence. While EGF-f1 corresponds to the native EGF-14 repeat, the others are frame-shifted EGF repeats. We carried out the oxidative folding of these peptides in vitro, analyzed the reaction mixtures by acid trapping followed by LC-MS, and isolated some of the resulting products. The oxidative folding of the native EGF-14 peptide is fast, produces a single three-disulfide species with an EGF-like disulfide topology and a marked difference in the RP-HPLC retention time compared with the starting product. On the contrary, frame-shifted peptides fold more slowly and give mixtures of three-disulfide species displaying RP-HPLC retention times that are closer to those of the reduced peptides. In contrast to the native EGF-14, the three-disulfide products that could be isolated are mainly unstructured, as determined by CD and NMR spectroscopy. We conclude that both kinetics and thermodynamics drive the correct pairing of cysteines, and speculate about how cysteine mispairing could trigger disulfide reshuffling in vivo.
Zanuttin
Francesco
F
International Centre for Genetic Engineering and Biotechnology, Protein Structure and Bioinformatics Group, Trieste, Italy.
Guarnaccia
Corrado
C
Pintar
Alessandro
A
Pongor
Sándor
S
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010100:Oxygen / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D019063:Tenascin / Q000737:chemistry*; D013816:Thermodynamics; D013997:Time Factors
D004220:Disulfides; D010455:Peptides; D019063:Tenascin; D004815:Epidermal Growth Factor; D003545:Cysteine; D010100:Oxygen