Contribution of tryptophan residues to the CD spectrum of the extracellular domain of human tissue factor

Abstract

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.

Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation

“…The circular dichroism spectrum of sTF thus obtained closely resembled the spectra previously obtained for sTF (43). The contents of secondary structure elements after deconvolution of the spectrum (spectral region 200-260 nm: 7.6% [α-helical], 28.7% [antiparallel β-sheet], 4.5% [parallel β-sheet], 28.2% [β-turn], and 39.0% [random coil]) were similar to those deduced from the x-ray structure of TF and to previously published work (43). DTT (1 mM) did not modify the secondary structure of sTF.…”

On the analysis of microrheological responses of self‐assembling RADA16‐I peptide hydrogel

“…With regard to the evident change in the CD spectra in blood serum milieu compared water, it can be said that in contact with the blood serum, conformation of the peptide changes. In Figure (B), it is clear that in the presence of blood serum, there are no considerable absorption band corresponding to β‐sheet structure; the CD spectra is around the 240 nm wavelength that represent the tertiary structure of proteins and peptides . The existence of absorption bands at 238 nm in the CD spectrum has been reported previously due to the neutralization of charged groups or the presence of salt .…”

Interaction of cyclic and linear Labaditin peptides with anionic and zwitterionic micelles

“…3B) took place. This alteration probably resulted from L 1 -micelle interaction that separated the W residues, culminating in the loss of the band at 225 nm [56,58,59] due to the L 1 conformational change.…”