| Autor: |
Khandekar, Sanjay S., Bettencourt, Brian M., Wyss, Daniel F., Naylor, Jerome W., Brauer, Pamela P., Huestis, Kevin, Dwyer, Donard S., Profy, Albert T., Osburne, Marcia S., Banerji, Julian, Jones, Barry |
| Zdroj: |
Journal of Biological Chemistry; December 1997, Vol. 272 Issue: 51 p32190-32197, 8p |
| Abstrakt: |
We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (Vα2Cα, Vβ8.2Cβ) expressed in insect cells binds both Vβ8.2-specific bacterial superantigen staphylococcal enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-Ak·conalbumin peptide complex with a low micromolar affinity. To define further the structural requirements for the TCR/ligand interactions, we have produced in Escherichia colia soluble, functional D10 single chain (sc) TCR molecule in which the Vα and Vβ domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-Ak·conalbumin peptide complex with thermodynamic and kinetic binding constants similar to those measured for the baculovirus-derived heterodimeric D10 TCR suggesting that neither the TCR constant domains nor potential N- or O-linked carbohydrate moieties are necessary for ligand recognition and for expression and proper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mm. Circular dichroism and NMR spectroscopy indicated that D10 scTCR is stabilized predominantly by β-sheet secondary structure, consistent with its native-like conformation. Because of its limited size, high solubility, and structural integrity, purified D10 scTCR appears to be suitable for structural studies by multidimensional NMR spectroscopy. |
| Databáze: |
Supplemental Index |
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