Notes on Detection of rare Partially folded molecules in equilibrium with the native conformation of RNaseH


RNase H*: E. coli ribonuclease H with all three free cysteines replaced by alanines



From Wikipedia:

The enzyme RNase H is a non-specific endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism. Members of the RNase H family can be found in nearly all organisms, from archaea and prokaryota and eukaryota.

RNase H’s ribonuclease activity cleaves the 3’-O-P bond of RNA in a DNA/RNA duplex to produce 3’-hydroxyl and 5‘-phosphate terminated products. In DNA replication, RNase H is responsible for removing the RNA primer, allowing completion of the newly synthesized DNA.

The term molten globule (MG) was first coined by A. Wada and M Ohgushi in 1983. It was first found in cytochrome c, which conserves a native-like secondary structure content but without the tightly packed protein interior, under low pH and high salt concentration. For cytochrome c and some other proteins, it has been shown that the molten globule state is a "thermodynamic state" clearly different both from the native and the denatured state, demonstrating for the first time the existence of a third equilibirum (i.e., intermediate)state.

Challenge of characterizing partially folded molecules

Examination of amide hydrogen exchange

Hypothesis developed by Englander: kinetic folding pathways of proteins can be deduced by determining the structure and stability of partially folded conformations under native conditions.

  • Behavior of how a protein folds can be inferred from the conformations of intermediate structures of the protein

Folding kinetics of RNaseH are known

Measurements for 53 amide protons, at 12 different denaturant concentrations

Exchanged measured as a function of time ranging from hours to months

Slower exchange than in unfolded polypeptide

 Addition of guanidine (denaturant) decreases protein stability, open form increases

  • Hydrogen exchange rates give info about structural transitions allowing that H exchange

Local fluctuations allow exchange without denaturant sensitivity m=0

Opening events dominating hydrogen exchange depend on denaturant conc. and amide proton being considered

RNase H unfolds in 3 regions

Blue=more stable 10kcal/mol avg, green region=8.7kcal/mol,


Circular Dichroism- differential absorption of left and right circularly polarized light.[2][3] UV CD is used to investigate the secondary structure of proteins


Therefore, the alpha helix of proteins and the double helix of nucleic acids have CD spectral signatures representative of their structures. The capacity of CD to give a representative structural signature makes it a powerful tool in modern biochemistry with applications that can be found in virtually every field of study.

Measuring exchange rates as function of denaturant reveals information pertaining to partial unfolding events or local flunctuations present at equilibrium

Helices are very stable interactions

Mutually stabilizing helices contribute more to stability than do beta sheets

Identify minima in energy landscape

Structure of one intermediate resembles molten globule occurring at low pH

  • Molten globules can model intermediates in folding pathways

Residues responsible for stability are also responsible for folding dynamics