SYNTHESIS, 3-DIMENSIONAL STRUCTURE, AND SPECIFIC N-15-LABELING OF THESTREPTOCOCCAL PROTEIN-G B1-DOMAIN

The 55-amino-acid B1-domain of the streptococcal protein G shows a hig h binding affinity to Ige isolated from a wide range of mammalian spec ies. Since the B1-domain forms an extremely stable globular folding un it containing the major secondary structure elements and is devoid of proline residues and disulfide bridges, it is also a useful tool for p rotein folding and stability studies. Its small size makes this protei n an ideal candidate for production by chemical synthesis, allowing in corporation of nan-natural amino acids with the possibility of assessi ng the influence of such residues on both the functional and structura l characteristics of proteins. In this study, we enployed three succes sive chemical syntheses of the B1-domain in order to define the optima l conditions of coupling and protection. The stepwise solid-phase meth odology using the tert-butyloxycarbonyl/benzyl strategy was used for t his purpose. First, the sequence assembly difficulties were evaluated. After analyzing of the problems found during assembly, a second optim ized synthesis was performed leading to formation of a synthetic B1-do main with a higher yield; the synthetic B1-domain was completely funct ional in its binding properties to IgG. Three orthogonal purification steps (gel-permeation, reverse-phase and ion-exchange HPLC) were requi red to obtain a sample suitable for structural analysis by high-resolu tion NMR. This study led to the conclusion that the synthetic B1-domai n adopts a three-dimensional structure identical to that of the molecu le obtained by recombinant techniques [Gronenborn, A. M., Filpula, D. R., Essig, N. Z., Achari, A., Whitlow, M., Wingfield, P. T. and Clore, G. M. (1991) Science 253, 657-661]. To demonstrate the usefulness of the chemical approach for the specific introduction of labelled amino acids in the primary structure, fourteen alpha-N-15-labelled amino aci ds were incorporated at selected critical positions during the third s ynthesis. This analog is the first in a series of molecules planned to study in detail the folding dynamics of the B1-domain.