In particular, AEDANS probes, attached to different positions, served as Fo��rster resonance energy transfer acceptors in combination with the single tryptophan residue Trp31 as FRET donor. In the native state, the endogenous Trp31 of CMPK is located at a surface exposed cavity that is formed by the N-terminal subdomain of the CORE-domain and Pro124. Our data suggest that folding of CMPK is highly concerted and involves at least one intermediate state with considerable secondary structure as opposed to UMPK where most secondary structure is only formed upon reaching the native state. Even more striking is the high kinetic stabilization that slows down unfolding around 100-fold compared to other NMPKs. The unfolding of CMPK in urea concentrations above 3.8 M is characterized by a single unfolding phase whose apparent rate constant ) increases exponentially with increasing concentrations of urea. The corresponding amplitude ) accounts for the total signal change indicating that there is no burst-phase. The refolding kinetics of CMPK could be determined between 0.6 M and 2.7 M urea. The rate constant for the fast phase lF1 is almost independent of the denaturant concentration for urea concentrations below 2.0 M. An increase at higher urea concentrations to values around 8 s21 can be observed. The slow phase lF3 decreases with decreasing amounts of urea between 0.6 and 2.0 M urea. Since rate constants in the range of 0.001�C0.1 s21 are indicative for Xaa-Pro bond isomerization processes, lF3 is most likely linked to prolyl-bond isomerization. lF1 deviates from the typical linear dependency on the denaturant concentration. This deviation could suggest that an intermediate is present in the folding mechanism. Especially the increase in lF1 with urea concentration is unusual for refolding reactions. Similar observations have been made for UMPK with increases in l1 and l2. Both cases can be related to theoretical considerations by Wildegger and Kiefhaber on folding of lysozyme who explain such behavior by the presence of a fast folding off-pathway intermediate that has to be unfolded before the next folding transition. In conjunction with the PI-103 chevron plot, the amplitude plot reveals lF3 as the main folding phase. Over the entire concentration range in the refolding experiments, AF3 stays almost constant with average amplitudes of 0.6 a.u. On the other hand AF1 is strongly dependent on the denaturant concentration. AF1 decreases between 0.6 and 2.7 M urea and the amplitude turns negative at 2.0 M urea. Interestingly, the change of amplitude AF1 coincides with the rollover of the according rate constant lF1 observed in the chevron plot. To detect a possible burst-phase in the folding or unfolding reaction of CMPK, the initial and final signals of the different measurements were plotted against the respective urea concentration. A deviation of the initial kinetic values from the baseline of the according BAY-60-7550 equilibrium values is an indication of a signal change within the dead-time of the stopped-flow. This deviation can be observed in the refolding process of CMPK, where initial refolding data differs from unfolded equilibrium data by a significant increase in signal amplitude. A possible explanation for such a fast process could be a rapid formation of a folding intermediate from which the native structure is formed. terrupted Unfolding Reveals Additional Folding Phase To further investigate the proline cis-trans isomerization in the unfolded state, the double-mixing technique was used. In contrast to the single mixing experiments described above, two mixing steps were applied. A possible picture that emerges from these considerations is that a folding burst leads to formation of a central core region containing Ala197 and generation of secondary structure elements.