Antibody heavy chain variable domains (VH) lacking their light chain (VL) 
partner are prime candidates for the design of minimum-size immunoreagents. To 
obtain structural information about isolated VH domains, a human VH was labelled 
with 15N or doubly labelled with both 15N and 13C and was studied by 
heteronuclear nuclear magnetic resonance spectroscopy. Most (90%) of the 1H and 
15N main-chain signals were assigned through two-dimensional TOCSY and NOESY 
experiments on the unlabelled VH and three-dimensional heteronuclear multiple 
quantum correlation TOCSY and NOESY experiments on the 15N-labelled VH. Four 
short stretches of the polypeptide chain could only be assigned on the basis of 
three-dimensional HNCA and HN(CO)CA experiments on the 13C-/15N-labelled 
protein. Long-range interstrand backbone NOEs suggest the presence of two 
adjacent beta-sheets formed by altogether nine antiparallel beta-strands. 3JNHC 
alpha H coupling constants and the location of slowly exchanging backbone amides 
support this interpretation. The secondary structure of the isolated VH is 
identical to that of heavy chain variable domains in intact antibodies, where VH 
domains are packed against a VL domain. The backbone assignments of the VH made 
it possible to locate its Protein A binding site. Chemical shift movements after 
complexing with the IgG binding fragment of Protein A indicate binding through 
one of the two beta-sheets of the VH. This beta-sheet is solvent exposed in 
intact antibodies. The Protein A binding site obviously differs from that on the 
Fc portion of immunoglobulins and is unique to members of the human VHIII gene 
subgroup.