Structural stability and internal motions of Escherichia coli ribonuclease HI: 15N relaxation and hydrogen-deuterium exchange analyses

J Mol Biol. 1998 Apr 3;277(3):707-22. doi: 10.1006/jmbi.1997.1622.


The relationship between the structural stability and the internal motions of proteins was investigated through measurements of 15N relaxation and hydrogen-deuterium exchange rates of ribonuclease HI from Escherichia coli and its thermostable quintuple mutant (Gly23-->Ala, His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His), which has a higher melting temperature by 20.2 degreesC. For most of the residues, the generalized order parameters (S2) obtained from 15N relaxation analyses as well as the localized hydrogen-bond-breaking motions (local breathing) observed as fast H-D exchange rates were largely unaffected by the mutations, indicating no global mutational effect on the internal motions. Several local mutational effects were observed for residues close to the mutation sites as follows. The S2 value significantly increased for Lys96 and Val98, which indicated that motions on the pico- to nanosecond time-scale became restricted within a protruding region including the Lys95-->Gly mutation site. In contrast, slight decreases in S2, and drastic increases in the chemical exchange motion on the micro- to millisecond time-scale (Deltaex), were observed for residues located in the joining region between the protrusion and the major domain of the protein. These changes may be caused by the elimination of the bulky Lys95 side-chain at the center of the protrusion. Deltaex observed for residues in alpha-helix I of the wild-type protein was reduced for the mutant, probably because a cavity in the hydrophobic core is filled by the Val74-->Leu mutation. The local breathing at position 134 was restricted by the Asp134-->His mutation, probably because the reduction of the negative charge repulsion contributes to the stability of the native major conformation relative to the breathing conformations around position 134.

MeSH terms

  • Amides
  • Deuterium
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Glycine / chemistry
  • Glycine / genetics
  • Hydrogen
  • Hydrogen Bonding
  • Lysine / chemistry
  • Lysine / genetics
  • Mutagenesis, Site-Directed
  • Nitrogen Isotopes
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Protein Conformation
  • Ribonuclease H / chemistry*
  • Ribonuclease H / genetics
  • Time Factors


  • Amides
  • Nitrogen Isotopes
  • Hydrogen
  • Deuterium
  • Ribonuclease H
  • ribonuclease HI
  • Lysine
  • Glycine