A T-CELL RECEPTOR V-ALPHA DOMAIN EXPRESSED IN BACTERIA - DOES IT DIMERIZE IN SOLUTION

To evaluate the potential for dimerization through a particular T cell receptor (TCR) domain, we have cloned the cDNA encoding a TCR V alpha from a hybridoma with specificity for the human immunodeficiency viru s (HIV) envelope glycoprotein 120-derived peptide P18-I10 (RGPGRAFVTI) bound to the murine major histocompatibility complex (MHC) class I mo lecule, H-2D(d). This cDNA was then expressed in a bacterial vector, a nd protein, as inclusion bodies, was solubilized, refolded, and purifi ed to homogeneity. Yield of the refolded material was from 10 to 50 mg per liter of bacterial culture, the protein was soluble at concentrat ions as high as 25 mg/ml, and it retained a high level of reactivity w ith an anti-V alpha 2 monoclonal antibody. This domain was monomeric b oth by size exclusion gel chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Circular dichroism spectra indica ted that the folded V alpha domain had secondary structure similar to that of single immunoglobulin or TCR domains, consisting largely of be ta sheet. Conditions for crystallization were established, and at leas t two crystal geometries were observed: hexagonal bipyramids that fail ed to diffract beyond similar to 6 Angstrom, and orthorhombic crystals that diffracted to 2.5 Angstrom. The dimerization of the V alpha doma in was investigated further by solution nuclear magnetic resonance spe ctroscopy, which indicated that dimeric and monomeric forms of the pro tein were about equally populated at a concentration of 1 mM. Thus, mo dels of TCR-mediated T cell activation that invoke TCR dimerization mu st consider that some V alpha domains have little tendency to form hom odimers or multimers.