ORIGIN OF DIMERIC STRUCTURE IN THE RIBONUCLEASE SUPERFAMILY

To enable application of postgenomic evolutionary approaches to unders tand the divergence of behavior and function in ribonucleases (RNases) , the impact of divergent sequence on the divergence of tertiary and q uaternary structure is analyzed in bovine pancreatic and seminal ribon ucleases, which differ by 23 amino acids, In a crystal, seminal RNase is a homodimer joined by two ''antiparallel'' intersubunit disulfide b onds between Cys-31 from one subunit and Cys-32' from the other and ha ving composite active sites arising from the ''swap'' of residues 1-20 from each subunit. Specialized Edman degradation techniques have comp leted the structural characterization of the dimer hi solution, new cr osslinking methods have been developed to assess the swap, and sequenc e determinants of quaternary structure have been explored by protein e ngineering using the reconstructed evolutionary history of the protein family as a guide. A single Cys at either position 32 (the first to b e introduced during the divergent evolution of the family) or 31 conve rts monomeric RNase A into a dimer. Even with an additional Phe at pos ition 31, another residue introduced early in the seminal lineage, swa p is minimal, A hydrophobic contact formed by Leu-28, however, also in troduced early in the seminal lineage, increases the amount of ''antip arallel'' connectivity of the two subunits and facilitates swapping of residues 1-20. Efficient swapping requires addition of a Pro at posit ion 19, a residue also introduced early in the divergent evolution of the seminal RNase gene. Additional cysteines required for dimer format ion are found to slow refolding of the protein through formation of in correct disulfide bonds, suggesting a paradox in the biosynthesis of t he protein. Further studies showed that the dimeric form of seminal RN ase known in the crystal is not the only form in vivo, where a substan tial amount of heterodimer is known, These data complete the acquisiti on of the background needed to understand the evolution of new structu re, behavior, and function in the seminal RNase family of proteins.