Contribution of tryptophan residues to the CD spectrum of the extracellular domain of human tissue factor : Application in folding studies and prediction of secondary structure

• The contribution to the circular dichroism (CD) spectrum made by each of the four Trp residues in the extracellular domain of human tissue factor, sTF (s designates soluble), was determined from difference CD spectra. The individual Trp CD spectra showed that all four residues contributed to the CD spectrum in almost the entire wavelength region investigated (180-305 nm). The sum of the individual spectra of each Trp residue in the near-UV region was qualitatively identical to the wild-type spectrum, clearly demonstrating that the Trp residues are the major contributors to the spectrum in this wavelength region. Trp CD bands interfere with the peptide bands in the far-UV region, leading to uncertainty in the predictions of the amounts of various types of secondary structure. Accordingly, the best prediction of secondary sTF structure content was achieved using a hypothetical Trp-free CD spectrum obtained after subtraction of all individual Trp spectra from the wild-type spectrum. The mutated Trp residues were also exploited as intrinsic probes to monitor the formation of local native-like tertiary structure by kinetic near-UV CD measurements. The global folding reaction was followed in parallel with a novel functional assay that registered the recovery of cofactor activity, i.e. stimulation of the amidolytic activity of Factor VIIa. From these measurements, it was found that sTF appears to regain FVIIa cofactor activity before the final side-chain packing of the Trp residues. The combined kinetic refolding results suggest that the compact asymmetric environments of the individual Trp residues in sTF are formed simultaneously, leading to the conclusion that the native tertiary structure of the whole protein is formed in a cooperative manner.

Nyckelord

Aromatic residues

Circular dichroism

Folding

Prediction

Secondary structure

Tissue factor

TECHNOLOGY

TEKNIKVETENSKAP

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