Non-native structure appears in microseconds during the folding of E. coli RNase H

The folding pathway of Escherichia coli RNase H is one of the best experimentally characterized for any protein. In spite of this, spectroscopic studies have never captured the earliest events. Using continuous-flow microfluidic mixing, we have now observed the first several milliseconds of folding by monitoring the tryptophan fluorescence lifetime (60 μs dead time). Two folding intermediates are observed, the second of which is the previously characterized I(core) millisecond intermediate. The new earlier intermediate is likely on-pathway and appears to have long-range non-native structure, providing a rare example of such non-native structure formation in a folding pathway. The tryptophan fluorescence lifetimes also suggest a deviation from native packing in the second intermediate, I(core). Similar results from a fragment of RNase H demonstrate that only half of the protein is significantly involved in this early structure formation. These studies give us a view of the formation of tertiary structure on the folding pathway, which complements previous hydrogen-exchange studies that monitored only secondary structure and observed sequential native structure formation. Our results provide detailed folding information on both a timescale and a size-scale accessible to all-atom molecular dynamics simulations of protein folding.