Estimation of the compaction of the denatured state by a protein variant involved in a reverse hydrophobic effect

Fluorescence resonance energy transfer methods have been used to evaluate changes in the dimension of the denatured state for position 73 variants of iso-1-cytochrome c that show a reverse hydrophobic effect [Herrmann et al. (1995)]. The experiments take advantage of the Trp 59/heme donor-acceptor pair in cytochrome c. Two large aliphatic variants, Ile 73 and Leu 73, were compared directly to the wild-type protein (lysine 73). The Leu 73 was an outlier in the original work and serves as an internal control. The data show that the volume of the denatured state is contracted by a small but significant degree, 4-6%, for the Ile 73 variant whereas the Leu 73, which does not conform to the reverse hydrophobic effect, shows no significant compaction. Given that position 73 is beyond Trp 59 in the sequence, the denatured state compaction appears to be a global effect.