NATIVE-LIKE IN-VIVO FOLDING OF A CIRCULARLY PERMUTED JELLYROLL PROTEIN SHOWN BY CRYSTAL-STRUCTURE ANALYSIS

A jellyroll beta-sandwich protein, the Bacillus beta-glucanase H(A16-M ), is used to probe the role of N-terminal peptide regions in protein folding in vivo. A gene encoding H(A16-M) is rearranged to place resid ues 1-58 of the protein behind a signal peptide and residues 59-214. T he rearranged gene is expressed in Escherichia coli. The resultant cir cularly permuted protein, cpA16M-59, is secreted into the periplasm, c orrectly processed, and folded into a stable and active enzyme. Crysta l structure analysis at 2.0-Angstrom resolution, R = 15.3%, shows cpA1 6M-59 to have a three-dimensional structure nearly identical with that of the parent beta-glucanase. An analogous experiment based on the wi ld-type Bacillus macerans beta-glucanase, giving rise to the circularl y permuted variant cpMAC-57, yields the same results. Folding of these proteins, therefore, is not a vectorial process depending on the conf ormation adopted by their native N-terminal oligopeptides after riboso mal synthesis and translocation through the cytoplasmic membrane.