CONFORMATIONAL INTEGRITY AND LIGAND-BINDING PROPERTIES OF A SINGLE-CHAIN T-CELL RECEPTOR EXPRESSED IN ESCHERICHIA-COLI

We recently showed that a soluble, heterodimeric murine D10 T-cell rec eptor (TCR) (V alpha 2C alpha, V beta 8.2C beta) expressed in insect c ells binds both V beta 8.2-specific bacterial superantigen staphylococ cal enterotoxin C2 (SECB) and a soluble, heterodimeric major histocomp atibility complex class II I-A(k).conalbumin peptide complex with a lo w micromolar affinity, To define further the structural requirements f or the TCR/ligand interactions, we have produced in Escherichia coli a soluble, functional D10 single chain (sc) TCR molecule in which the V alpha and V beta domains are connected by a flexible peptide linker, Purified and refolded D10 scTCR bound to SEC2 and murine major histoco mpatibility complex class II I-A(k).conalbumin peptide complex with th ermodynamic and kinetic binding constants similar to those measured fo r the baculovirus-derived heterodimeric D10 TCR suggesting that neithe r the TCR constant domains nor potential N- or O-linked carbohydrate m oieties are necessary for ligand recognition and for expression and pr oper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mM. Circular dichroism and NMR spectroscopy ind icated that D10 scTCR is stabilized predominantly by beta-sheet second ary structure, consistent with its native-like conformation. Because o f its limited size, high solubility, and structural integrity, purifie d D10 scTCR appears to be suitable for structural studies by multidime nsional NMR spectroscopy.