SECRETION EFFICIENCY IN SACCHAROMYCES-CEREVISIAE OF BOVINE PANCREATICTRYPSIN-INHIBITOR MUTANTS LACKING DISULFIDE BONDS IS CORRELATED WITH THERMODYNAMIC STABILITY

Bovine pancreatic trypsin inhibitor (BPTI) has been widely used as a m odel protein to investigate protein structure and folding pathways. To study the role of its three disulfide bonds in folding, proofreading, and secretion of BPTI in an intact eucaryotic cell, BPTI was expresse d and secreted from a synthetic gene in the yeast Saccharomyces cerevi siae. Site-directed mutagenesis was used to create all possible single and pairwise cysteine to alanine BPTI mutants, and the effect of thes e mutations on secretion efficiency was determined. The 5-55 disulfide bond is found to be essential for secretion-loss of either Cys5, Cys5 5, or both prevents secretion. Removal of the 14-38 disulfide bond res ults in a small reduction of secretion, but individual Cys14 or Cys38 replacements reduce secretion efficiency by 30%. Cys30 and Cys30-51 mu tants are secreted at half the level of wild-type BPTI, while secretio n of the Cys51 mutant is reduced by 90%. BPTI containing only a single disulfide bond (5-55) is not secreted. No relationship is observed be tween secretion efficiency and in vitro folding or unfolding rates, bu t mutant BPTI secretion is directly correlated with the in vitro unfol ding temperature T-m and the free energy of stabilization provided by each of the three disulfides. These results indicate that structural f luctuations rather than the time-averaged structure observed by NMR or X-ray crystallography may determine recognition of a protein as misfo lded and subsequent retention and degradation.