Detection of altered protein conformations in living cells

The maturation, conformational stability, and the rate of in vivo degradati on are specific for each protein and depend on both the intrinsic features of the protein and those of the surrounding cellular environment. While syn thesis and degradation can be measured in living cells, stability and matur ation of proteins are more difficult to quantify. We developed the split-ub iquitin method into a tool for detecting and analyzing changes in protein c onformation. The biophysical parameter that forms the basis of these measur ements is the time-averaged distance between the N terminus and C terminus of a protein. Starting from three proteins of known structure, we demonstra te the feasibility of this approach, and employ it to elucidate the effect of a previously described mutation in the protein Sec62p on its conformatio n in Living cells. (C) 2001 Academic Press.