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 virus (HIV) envelope glycoprotein 120-derived peptide P18-110 (RGPGRAFVTI) bound to the murine major histocompatibility complex (MHC) class I molecule, H-2Dd. This cDNA was then expressed in a bacterial vector, and protein, as inclusion bodies, was solubilized, refolded, and purified to homogeneity. Yield of the refolded material was from 10 to 50 mg per liter of bacterial culture, the protein was soluble at concentrations as high as 25 mg/ml, and it retained a high level of reactivity with an anti-V alpha 2 monoclonal antibody. This domain was monomeric both by size exclusion gel chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Circular dichroism spectra indicated that the folded V alpha domain had secondary structure similar to that of single immunoglobulin or TCR domains, consisting largely of beta sheet. Conditions for crystallization were established, and at least two crystal geometries were observed: hexagonal bipyramids that failed to diffract beyond approximately 6 A, and orthorhombic crystals that diffracted to 2.5 A. The dimerization of the V alpha domain was investigated further by solution nuclear magnetic resonance spectroscopy, which indicated that dimeric and monomeric forms of the protein were about equally populated at a concentration of 1 mM. Thus, models of TCR-mediated T cell activation that invoke TCR dimerization must consider that some V alpha domains have little tendency to form homodimers or multimers.
Plaksin
D
D
Molecular Biology Section, National Institute of Allergy and Infections Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Chacko
S
S
McPhie
P
P
Bax
A
A
Padlan
E A
EA
Margulies
D H
DH
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins