HYDROGEN-EXCHANGE STUDIES OF THE ARC REPRESSOR - EVIDENCE FOR A MONOMERIC FOLDING INTERMEDIATE

The hydrogen exchange rates of the backbone amide and labile side-chai n protons of the dimeric Arc repressor have been measured. For the slo wly exchanging amides in the alpha-helical regions, these rates show a concentration dependence. To account for this dependence, the role of the monomer-dimer equilibrium was considered. Extrapolating the obser ved exchange rates to zero dimer concentration provides estimates of t hese rates in the monomer and shows that they are significantly retard ed compared to those of an unfolded polypeptide. This suggests that th e monomer is in a structured ''molten globule'' like state. In particu lar, the two helices of Arc retain a high degree of their secondary st ructure and it is proposed that the two amphiphilic helices are packed together with their hydrophobic faces. Evidence for a partially folde d structure in the Arc monomer was reported earlier in two other studi es [J. L. Silva, C. F. Silveira, A. Correia, Jr., and L. Pontes (1992) Journal of Molecular Biology, Vol. 223, pp. 545-555; X. Peng, J. Jona s, and J. L. Silva (1993) Proceedings of the National Academy of Scien ce USA, Vol. 90, pp. 1776-1780]. By combining the results of these stu dies and ours, a folding pathway of the dimeric Arc repressor involvin g four different stages is proposed. Due to the low concentration of A rc repressor in the cell, the protein is present either as a free mono mer or it is bound to DNA presumably as a tetramer. Therefore the fold ing pathway can be regarded as an integral part of the overall DNA bin ding process. (C) John Wiley & Sons, Inc.