Folding of epidermal growth factor-like repeats from human tenascin studied through a sequence frame-shift approach

Epidermal growth factor ( egf) domains are 30-50 residue long repeats characterized by the strict conservation of six cysteine residues, which are forming three disulfide bonds with the topology 1-3, 2-4, 5-6. The common structural feature of egf domains is a two-stranded beta-sheet from which the three disulfide bonds depart to connect the N- and C-tem1inal loops, to make a rather compact structure. Beside the six cysteines, a wide variability in the length and composition of the stretches connecting the cysteines has been observed. Probably because of its capability to accommodate very different sequences on a common scaffold, the egf domain is one of the most frequently employed building blocks in modular proteins. In order to investigate the factors that determine the correct folding of epidermal growth factor-like repeats ( egf) within a multi-domain protein, we prepared a series of six peptides that, taken together, span the sequence of two egf repeats of human tenascin, a large extracellular matrix glycoprotein expressed during embryonic development and in proliferative processes such as wound healing and tumorigenesis. 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-fl to egf-f6, that are 33 residue long, contain six cysteines each, and bear a partial overlap in the sequence. While egf-fl corresponds to the native egf-14 repeat, the others are frameshifted 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. TI1e 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 to the starting product. On the contrary, frame-shifted peptides fold more slowly and give mixtures of threedisulfide 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 m1Spamng could trigger disulfide reshuffling in viva.