Multiple time-scale dynamics of homologous thermophilic and mesophilic
ribonuclease HI enzymes

Residue numbering is different between PDB file 1RIL and current entry:
PDB entry 1ril is based on E. coli ribonuclease HI numbering (ie. first
ttRNH residue is Met -4), while this BMRB entry uses an independent
numbering system (ie. first ttRNH residue is Met 1). In addition, all
Cys residues have been replaced by Ala or Ser in this protein:
C17A, C45S, C67A, C154S.

Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes.

The mechanisms by which thermophilic proteins attain their increased
thermostability remain unclear, as usually the sequence and structure of these
proteins are very similar to those of their mesophilic homologues. To gain
insight into the basis of thermostability, we have determined protein stability
curves describing the temperature dependence of the free energy of unfolding
for two ribonucleases H, one from the mesophile Escherichia coli and one from
the thermophile Thermus thermophilus. The circular dichroism signal was
monitored as a function of temperature and guanidinium chloride concentration,
and the resulting free energies of unfolding were fit to the Gibbs-Helmholtz
equation to obtain a set of thermodynamic parameters for these proteins.
Although the maximal stabilities for these proteins occur at similar
temperatures, the heat capacity of unfolding for T. thermophilus RNase H is
lower, resulting in a smaller temperature dependence of the free energy of
unfolding and therefore a higher thermal melting temperature. In addition, the
stabilities of these proteins are similar at the optimal growth temperatures
for their respective organisms, suggesting that a balance of thermodynamic
stability and flexibility is important for function.

Hollien J, Marqusee S.
A thermodynamic comparison of mesophilic and thermophilic ribonucleases H.
Biochemistry. 1999 Mar 23;38(12):3831-6.

A thermodynamic comparison of mesophilic and thermophilic ribonucleases H.

Stability parameters for individual residues in Thermus thermophilus
cysteine-free RNase H were determined by native state hydrogen exchange, thus
providing a unique comparison of regional thermodynamics between thermophilic
and mesophilic homologues. The general distribution of stability in the
thermophilic protein is similar to that of its mesophilic homologue, with a
proportional increase in stability for almost all residues. As a consequence,
the residue-specific stabilities of the two proteins are remarkably similar
under conditions where their global stabilities are the same. These results
indicate that T. thermophilus RNase H is stabilized in a delocalized fashion,
preserving a finely tuned balance of stabilizing interactions throughout the
structure. Therefore, although protein stability can be altered by single amino
acid substitution, evolution for optimal function may require more subtle and
delocalized mechanisms.

Hollien J, Marqusee S.
Structural distribution of stability in a thermophilic enzyme.
Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):13674-8.

Proceedings of the National Academy of Sciences of the United States of America
Structural distribution of stability in a thermophilic enzyme.

Crystal Structure Of Ribonuclease H From Thermus Thermophilus Hb8 Refined At 2.8 Angstroms Resolution

ribonuclease H (RNase H) [Thermus thermophilus HB8]

ribonuclease h [Thermus thermophilus HB8]

ribonuclease H (EC 3.1.26.4) - Thermus aquaticus

RecName: Full=Ribonuclease H; Short=RNase H [Thermus thermophilus HB8]

Plasmid used for overexpression was obtaind from Dr. Susan Marqusee: a
pAED4 vector with synthesized rnhA gene that has codons optimized for
expression in E. coli (see Ref. 1).

All 1H and 15N resonances reported are for the TROSY peak at 14.1 T.