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

Proc Natl Acad Sci U S A. 2014 Oct 14;111(41):14746-51. doi: 10.1073/pnas.1410630111. Epub 2014 Sep 25.

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 rate-limiting 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acids / metabolism*
  • Anilino Naphthalenesulfonates / metabolism
  • Circular Dichroism
  • DNA Mutational Analysis
  • Enzyme Stability / drug effects
  • Escherichia coli / enzymology*
  • Fluorescence
  • Hydrophobic and Hydrophilic Interactions / drug effects
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Mutation / genetics
  • Protein Folding* / drug effects
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Ribonuclease H / chemistry*
  • Ribonuclease H / genetics
  • Ribonuclease H / metabolism*
  • Urea / pharmacology

Substances

  • Amino Acids
  • Anilino Naphthalenesulfonates
  • 1-anilino-8-naphthalenesulfonate
  • Urea
  • Ribonuclease H