Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule

Abstract

The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the ratelimiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates.

Evolutionary trend toward kinetic stability in the folding trajectory of RNases H

“…In addition, the robust folding of the RNase H ancestors is consistent with evidence indicating that ASR generates proteins of evolutionary significance, unlike consensus proteins, which, despite being likened to ancestral proteins, have been found to fold poorly in some instances (18,51). Previous work on extant RNases H has established that the "core" region of the protein (helices A-D and strands 4 and 5) folds early and is structured in the intermediate, whereas the periphery (helix E and strands 1-3) remains unfolded until the ratelimiting step (23,(52)(53)(54) (Fig. S5).…”

Non-Native Structure Appears in Microseconds during the Folding of E. coli RNase H

“…Globally fitting the average fluorescence lifetime together with total fluorescence intensity (Figure 6b – inset) to a two-state model yields a stability of 1.0 ± 1.0 kcal/mol and an m-value of 0.5 ± 0.2 kcal/mol/M urea ; however, improvements in χ 2 using a three-state model hint that the fragment may populate an intermediate during equilibrium unfolding. Three-state equilibrium unfolding by fluorescence would explain why the stability and m-value determined here using a two-state model are lower than previously determined using CD data fit to a two-state model (stability of 3.0 ± 0.3 kcal/mol and m-value of 1.16 ± 0.05 kcal/mol/M urea ) [9]. Nevertheless, the equilibrium average lifetime data are useful to compare to the kinetic amplitudes.…”

“…Since there are no tryptophans in this region of the protein, our kinetic experiments do not report on its role in the early folding pathway. However, we recently made a fragment of RNase H with the I–III/E regions removed [9]. This fragment folds and serves as a mimic of I core .…”

“…Kinetic modeling, mutational analysis, and comparison with the HX-MS data [7] suggest that I early is an on-pathway intermediate containing some non-native structure. Using a fragment of RNase H [9], we confirm that only half the protein is significantly involved in these early folding steps. These results, together with the previous HX-MS data [7], provide a detailed model for the early folding of RNase H on both a timescale and size-scale amenable to comparison with atomistic folding simulations.…”

“…In the native state, most of the tryptophans have some solvent exposure (Figure 1b,c) and all are packed against other tryptophans, both of which are undoubtedly the basis of the observed quenching. Previous studies using HX [2,7], mutagenesis [6] and equilibrium mimics [9] all suggest a native-like topology for I core . However, since all of the tryptophans reside within the structured region of I core , the non-native fluorescence signal of I core suggests that it contains some non-native features.…”