Fluctuations of primary ubiquitin folding intermediates in a force clamp.

Folding experiments of single ubiquitin molecules under force clamp using an atomic force microscope revealed a dynamic long-lived intermediate with nanometer scale end-to-end distance fluctuations along an unexpectedly complex folding pathway. To examine the nature of this intermediate at the atomic level as well as the driving forces that give rise to the observed fluctuations, we performed molecular dynamics refolding simulations of unfolded ubiquitin under constant force. After an initial fast collapse, we find a highly dynamic, broad ensemble of conformations with partial and continuously changing secondary structure and side chain interactions. This ensemble resembles a molten-globule-like state, similar in nature to the previously described non-native state of ubiquitin in solution, but stretched by the external force. The scale of the end-to-end distance fluctuations derived from the simulations compares well with experiment. Transient formation of unspecific and metastable hydrophobic clusters along the chain are found to give rise to the observed end-to-end distance fluctuations. These distinct collapses, interpreted as folding attempts, imply an upper limit for the folding attempt frequency of approximately 10 ns. Our results suggest possible relations between force-induced unfolding and temperature or chemically induced denaturation.