After each heating cycle, the activity of individual enzyme molecules changed randomly from its prior activity, with approximately 50% of the population gaining activity and 50% losing activity. Considering the error associated with linear fitting, a maximum of 12% of the enzyme population may have Tetrahydroberberine overlapping activity values after each pulse. There are several possible results that may be expected to occur upon heating an enzyme. First, if the activation barrier is too high for the molecule to convert to a different local minimum, the enzyme will fall back into the same local minimum, maintain its original conformation, and there will be no change in the reaction rate after heating. Second, if the enzyme overcomes the energy barrier, it may convert to a new stable conformation. The new conformation may have either a faster or slower reaction rate. The changes in activity could also be a result of the tetramer-dimermonomer equilibrium. At the single molecule level, however, the tetramer-dimer-monomer equilibrium is shifted to the monomer form due to the low concentration of monomer in the microwells such that if the tetramer dissociates, we would expect an irreversible loss in enzyme activity��a phenomenon we observe for a small number of enzyme molecules in these experiments. Narrowing of the distribution after the first heating pulse may be caused by kinetic traps that have slightly higher energy barriers and are only accessible when the nascent protein folds; thus some enzymes cannot return to their previous states upon re-heating at 47uC. No correlation between the activities of enzymes before and after heating was observed, suggesting that enzymes have no ‘memory’ of any previous conformations before the heating pulse was introduced. The change in activity of each enzyme was random as well, i.e. it did not correlate with its initial activity. Each enzyme exhibits an equal probability of either gaining or losing activity through all heating periods. As temperature increases, a protein molecule is expected to gain enough energy to overcome an energy barrier and convert to a new conformation that has a different activity. If conformational differences between different enzyme molecules are the cause of the broad activity distribution, then the activities of the individual molecules should redistribute after each heating pulse, which is what we observe. On the other hand, if sequence differences are the sole basis for the activity distribution, then upon heating, each enzyme molecule should revert to its original activity. Our results clearly show that virtually all the enzyme molecules redistribute their activity when heated, demonstrating the importance of conformations in static heterogeneity. It is important to note that both conformation and sequence may contribute to the activity distribution because enzyme molecules with different sequences will also interconvert between conformations upon heating. Loganin Moreover, enzymes with different primary sequences may display varying kinetic responses upon heating. In addition to the observed change in activity due to conformational changes, denaturation of some enzyme molecules occurred, which is likely the reason for some loss in activity when activity is measured in bulk solution. These enzymes are irreversibly denatured and contribute to the decrease in average activity over time. The denaturation temperature of b-galactosidase was calculated to be 55uC by CD experiments and confirmed by kinetic measurements, and agrees well with denaturation temperatures reported in the literature. In the present experiments, denaturation of the enzymes was minimized by heating only to 47uC, which is below this denaturation temperature.