USE OF PROLINE MUTANTS TO HELP SOLVE THE NMR SOLUTION STRUCTURE OF TYPE-III ANTIFREEZE PROTEIN

To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a T ype III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP iso forms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by incl usion of an N-terminal methionine but was indistinguishable in activit y. The NMR spectra of this AFP were complicated by cis-trans proline i somerization from the C-terminal sequence YPPA. Substitution of this s equence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination u sing 2D H-1 NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparal lel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mut ants were valuable in clarifying ambiguous NMR spectral assignments am ongst the remaining six prolines of rQAE m1.1. In contrast to the repl acement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein so lubility and antifreeze activity.