Use of the phase diagram method to analyze the protein unfolding-refolding reactions: Fishing out the invisible intermediates

Partially folded conformations are important players in protein self-organization, function, and misfolding, thus attracting the intensive and constant attention of researchers. Different conformational states of proteins can be easily discriminated from one another using the multiparametric approach, which usually involves the application of several physicochemical methods sensitive to the various structural levels of a protein molecule. This approach gives the most complete information about the structural changes in protein during its de- and renaturation. However, in many cases, researches are interested in a brief and simple test for the appearance of an intermediate state. The binding of the hydrophobic fluorescent probe ANS represents one of the well-established techniques, allowing the retrieval of such information. We are introducing another approach, based on the analysis of the spectroscopic data in a form of spectral diagrams. The essence of this method is to build the l(lambda1) versus l(lambda2) dependence, where l(lambda1) and l(lambda2) are the spectral intensity values (e.g., fluorescence, CD, FTIR, absorbance, etc.) measured on wavelengths lambda(1) and lambda(2). We are showing that this approach gives the detailed description of the protein unfolding pathway, detects the numerous partially folded species, and even fishes out the hidden intermediates. An application of this method for the analysis of the equilibrium and kinetic unfolding/refolding of several proteins under the variety of conditions is described. Advantages and disadvantages of the technique suggested are also discussed.