Three factors determine the temperature range at which global unfolding free energies (Δ

*G*_{U}) can be determined by quantitative cysteine reactivity. The first limits are set by the accuracy of the measurement of the labeling rate constants: an upper limit occurs at a temperature (

) and free energy (

^{min}Δ

*G*_{exp}) at ∼10 °C above

*T*_{m} (red-dashed arrows) where the difference of

*k*_{label} and

*k*_{int} is within experimental error; a lower limit occurs at a temperature (

) and free energy (

^{max}Δ

*G*_{exp}) at ∼10–20 °C below

*T*_{m} (green-dashed arrows) where increased stability sufficiently reduces

*k*_{label} ( and ) such that it appears to be independent of temperature within experimental error. The second limit is set in some cases where the mechanism of cysteine protection (i.e. local or global unfolding) is dependent on temperature. Such cases manifest themselves as a deviation of the observed temperature dependence of Δ

*G*_{U} from that expected for global unfolding. It is well established that global unfolding conditions prevail within ∼10–20 °C of

*T*_{m} (, ), which we refer to as the global unfolding window of observation. The black line illustrates a case in which there is no such switch (modeled by ) and the GUWO extends over the entire temperature range; the gray line represents switching between global and local unfolding with a concomitant temperature limit for the GUWO (modeled by Eq. 12 of ref. ). The third limit is set at a point where EX1 conditions prevail and

*k*_{close} no longer exceeds

*k*_{int} (not illustrated). This may occur as stability is diminished (Δ

*G*_{U} < 1 kcal/mol) or if the concentration of thiol probe [

*P*] is too high. Loss of EX2 conditions is manifested as a loss of the linear dependence of

*k*_{label} on [

*P*] and can be remedied by reducing [

*P*]. The overall temperature range at which observations can be made is the intersection of all three of these conditions (black and gray bars).

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