Supplementary Figure 2: Schematic representation of possible unfolding scenarios.

Schematic temperature vs. fluorescence curves are shown, with different colors representing three phases of the entire unfolding transition. The left white part represents the folded phase, the middle orange part the unfolding phase and the right white part the aggregation phase. The half-maximal intensity corresponding to the inflection point of the curve and thus the melting temperature is depicted as dotted line. The dashed line curve insets represent an idealized two state unfolding behavior, with a steep transition. Cartoons of the behavior of proteins are depicted below the graph. (a) A typical unfolding transition curve of a single domain protein is shown. Note, that it overlays well with the dashed line and therefore it is assumed to represent two-state unfolding. (b) The unfolding curve for an instable protein complex is shown. The complex disassembles before it unfolds, yielding multiple transitions and a considerable divergence from two-state unfolding. (c) The unfolding transition for another instable protein complex is shown. Disassembly and unfolding occur in a narrow temperature range yielding multiple transitions which superimpose in a way that they cannot be distinguished anymore. However, this yields a shallow transition in comparison to the dashed line curve (i.e. two-state unfolding). (d) The unfolding curve of a stable complex is shown. Disassembly and unfolding occur in a near concerted manner. Thus, the curve resembles the two-state transition shown in (a).