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.