title: Two-dimensional IR correlation spectroscopy: sequential events in the unfolding process of the lambda cro-V55C repressor protein.
abstract: 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 events. It seems that, at least in certain cases, two-dimensional (2D) IR correlation 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 intermediate is compatible with that of a partially unfolded protein and involves a reorganization of the N terminus, whereas the antiparallel beta-ribbon formed by the C-terminal part of each subunit remains largely intact. To establish whether the unfolding process involves sequential events, we have performed a 2D correlation analysis of IR spectra recorded over the temperature range of 20-95 degrees C. The 2D IR correlation analysis indeed provides evidence for a sequential formation of the stable intermediate, which is created 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-helices in a second step, and the third step comprises the reorganization of the remaining beta-sheet and of some unordered segments in the protein. The 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 beta-strands at the dimer interface.
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany. henry.mantsch@nrc.ca
authors.forename: H
authors.initials: H
authors.lastname: Fabian
authors.forename: H H
authors.initials: HH
authors.lastname: Mantsch
authors.forename: C P
authors.initials: CP
authors.lastname: Schultz
mesh_terms: D010582:Bacteriophage lambda / Q000737:chemistry*; D004268:DNA-Binding Proteins; D019281:Dimerization; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry*; D013055:Spectrophotometry, Infrared / Q000379:methods*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins