Two-dimensional IR correlation spectroscopy: Sequential events in the unfolding process of the lambda Cro-V55C repressor protein

A question often posed in protein folding/unfolding studies is whether the process is fully cooperative or whether it contains sequential elements. To address this question, one needs tools capable of resolving different even ts. It seems that, at least in certain cases, too-dimensional(2D) IR correl ation spectroscopy can provide answers to this question. To illustrate this point, we have turned to the Cro-V55C dimer of the lambda Cro repressor, a protein known to undergo thermal unfolding in two discrete steps through a stable equilibrium intermediate. The secondary structure of this intermedi ate is compatible with that of a partially unfolded protein and involves a reorganization of the N terminus, whereas the antiparallel beta-ribbon form ed by the C-terminal part of each subunit remains largely intact. To establ ish whether the unfolding process involves sequential events, we have perfo rmed a 2D correlation analysis of IR spectra recorded over the temperature range of 20-95 degrees C. The 2D IR correlation analysis indeed provides ev idence for a sequential formation of the stable intermediate, which is crea ted in three (closely related) steps. A first step entails the unfolding of the short N-terminal beta-strand, followed by the unfolding of the alpha-h elices in a second step, and the third step comprises the reorganization of the remaining beta-sheet and of some unordered segments in the protein. Th e complete unfolding of the stable intermediate at higher temperatures also undergoes sequential events that ultimately end with the breaking of the H bonds between the two a-strands at the dimer interface.