Detection of rare partially folded molecules in equilibrium with the native conformation of RNaseH

  title={Detection of rare partially folded molecules in equilibrium with the native conformation of RNaseH},
  author={Aaron Keith Chamberlain and Tracy M. Handel and Susan Marqusee},
  journal={Nature Structural Biology},
Despite the general observation that single domain proteins denature in a completely cooperative manner, amide hydrogen exchange of ribonuclease H in low levels of denaturant demonstrates the existence of two partially folded species. The structures of these marginally stable species resemble kinetic folding intermediates and the molten globule state of the protein. These data suggest that the first region to fold is the thermodynamically most stable portion of the protein and that the molten… 
The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules detected under native conditions
Results indicate that the first portion of ribonuclease HI to fold is the most thermodynamically stable region of the native state, and that folding of this protein follows a hierarchical process.
Confirmation of the hierarchical folding of RNase H: a protein engineering study
Results indicate that interactions formed in the intermediate persist in the transition and native states and that RNase H folds through a hierarchical mechanism.
Folding of an isolated ribonuclease H core fragment
It is shown indirectly that the monomeric form of eABCD is folded and has an overall secondary structure similar to the dimeric form.
Native-state energetics of a thermostabilized variant of ribonuclease HI.
Though increased in stability by a uniform factor, D10A shows a distribution of stabilities in its secondary structural units that is similar to that of E. coli RNase H, but not the closely related protein from Thermus thermophilus, Hence, the simple mutation used to stabilize the enzyme does not recreate the balance of conformational flexibility evolved in the thermophilic protein.
The energetics of T4 lysozyme reveal a hierarchy of conformations
The overall subdomain hierarchy of energies does not mirror data on the folding pathway for this protein, challenging the relationship between energy landscapes and folding trajectories.
Hydrogen exchange studies of protein structure.


Protein folding intermediates: native-state hydrogen exchange.
The partially unfolded forms detected by hydrogen exchange appear to represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions and thus may define the major pathway for cytochrome c folding.
The barriers in protein folding
Elimination of an interaction which forms in denatured cytochrome c enables the majority of the molecules to fold to the native state on a 15 ms time scale, without populating observable
Structure of the acid state of Escherichia coli ribonuclease HI.
The residues that are responsible for the observed structure of the acid state of RNase H* are determined using amide hydrogen exchange carried out under acid state conditions, followed by quenching and NMR detection on the native state.
Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin.
Hydrogen exchange pulse labeling and stopped-flow circular dichroism were used to establish that the structure of the earliest detectable intermediate formed during refolding of apomyoglobin
Equilibrium unfolding of Escherichia coli ribonuclease H: Characterization of a partially folded state
The acid state of E. coli RNase H has the characteristics of a molten globule: it retains a high degree of secondary structure, remains compact, yet does not appear to contain a tightly packed core.
Hydrogen exchange and structural dynamics of proteins and nucleic acids.
Though the structures presented in crystallographic models of macromolecules appear to possess rock-like solidity, real proteins and nucleic acids are not particularly rigid. Most structural work to
Advances in spectroscopy, protein engineering, and peptide synthesis have had a dramatic impact on the understanding of the structures and stabilities of transient folding intermediates. The data
Guanidinium chloride induction of partial unfolding in amide proton exchange in RNase A.
The results indicate that these concentrations of GdmCl do induce exchange by means of a partial unfolding mechanism for all 23 protons; this implies that exchange reactions can be used to study the unfolding and stability of local regions.
Three-dimensional structure of ribonuclease H from E. coli
THE three-dimensional structure of RNase H from Escherichia coli was determined at 1.8 Å resolution by X-ray crystallography. The enzyme was found to belong to the α + β class of structures,
Hydrogen exchange in unligated and ligated staphylococcal nuclease.
Protection factors for the slowly exchanging hydrogens in unligated nuclease H124L at 37 degrees C and pH 5.5 were found to vary by over one order of magnitude, and exchange of these hydrogens thus appears to occur via global unfolding of the protein.