Remarkable Stabilization of a Psychrotrophic RNase HI by a Combination of Thermostabilizing Mutations Identified by the Suppressor Mutation Method

Abstract

Ribonuclease HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 (So-RNase HI) is much less stable than Escherichia coli RNase HI (Ec-RNase HI) by 22.4 degrees C in T m and 12.5 kJ mol (-1) in Delta G(H 2O), despite their high degrees of structural and functional similarity. To examine whether the stability of So-RNase HI increases to a level similar to that of Ec-RNase HI via introduction of several mutations, the mutations that stabilize So-RNase HI were identified by the suppressor mutation method and combined. So-RNase HI and its variant with a C-terminal four-residue truncation (154-RNase HI) complemented the RNase H-dependent temperature-sensitive (ts) growth phenotype of E. coli strain MIC3001, while 153-RNase HI with a five-residue truncation could not. Analyses of the activity and stability of these truncated proteins suggest that 153-RNase HI is nonfunctional in vivo because of a great decrease in stability. Random mutagenesis of 153-RNase HI using error-prone PCR, followed by screening for the revertants, allowed us to identify six single suppressor mutations that make 153-RNase HI functional in vivo. Four of them markedly increased the stability of the wild-type protein by 3.6-6.7 degrees C in T m and 1.7-5.2 kJ mol (-1) in Delta G(H 2O). The effects of these mutations were nearly additive, and combination of these mutations increased protein stability by 18.7 degrees C in T m and 12.2 kJ mol (-1) in Delta G(H 2O). These results suggest that several residues are not optimal for the stability of So-RNase HI, and their replacement with other residues strikingly increases it to a level similar to that of the mesophilic counterpart.

Thermal Adaptation of Conformational Dynamics in Ribonuclease H

“…The handle loop has previously been suggested to move as a rigid body in ecRNH and ttRNH; these results suggest that it can either swing on loose hinges, or be buttressed by the sidechain-backbone hydrogen bonds for which an Asn residue at this site is uniquely well-suited. A suppressor screen for thermostabilizing mutations of soRNH, which natively contains Lys at this position, identified K90N as thermostabilizing by 0.7 kcal/mol with only a 9% decrease in activity relative to the wild-type protein [29], consistent with our observations by computational mutagenesis that these reciprocal mutations are mostly nondisruptive and are easily accommodated in the local environment. Interestingly, among bacterial proteins containing handle loops, the frequency of ocurrence of Asn is higher among those sequences annotated as having a thermophilic source organism than among those annotated as being derived from mesophiles (Figure S9).…”

“…Reciprocal mutations have identified five distinct sites that collectively contribute about half of this stability difference [28]. More recently, similar analyses have identified mutations that confer increased thermostability to the homolog from the psychrotrophic bacterium Shewanella oneidensis (soRNH) [29]; like many proteins from cold-tolerant organisms [30], soRNH is natively thermolabile compared to its mesophilic homolog. Furthermore, comparison of the thermodynamic parameters of ecRNH, ttRNH, and an additional homolog from the moderately thermophilic bacterium Chlorobium tepidum reveals that the more thermostable proteins share a common mechanism of stabilization in the form of increased values of , likely owing to the existence of residual structure in the unfolded state [31], [32].…”

Destabilization of psychrotrophic RNase HI in a localized fashion as revealed by mutational and X‐ray crystallographic analyses

“…Tyr24 and Phe41 are almost fully buried inside the protein molecule, whereas Gln149 is relatively well exposed to the solvent. We have shown previously that the Asp39 → Ala mutation also stabilizes the protein to a similar level as that of D39G‐RNase HI [13]. Asp39 is changed to Ala (Ala37) in Ec‐RNase HI, which is buried inside the protein molecule by 83%.…”

“…Both the hydrogen bond and ion pair have been reported to contribute to protein stabilization [16,17]. However, the finding that So‐RNase HI is stabilized by the Asn29 → Lys mutation by 3.6 °C in T m and 3.5 kJ·mol −1 in Δ G (H 2 O) [13] suggests that the stabilization effect caused by the introduction of an ion pair at the mutation site is stronger than the destabilization effect caused by the elimination of two hydrogen bonds at the same site. Several proteins have also been reported to be stabilized by the introduction of ion pairs [18–21].…”

Stabilization by Fusion to the C-terminus of Hyperthermophile Sulfolobus tokodaii RNase HI: A Possibility of Protein Stabilization Tag

“…Structure-based design [1], [2], sequence alignment approaches [3], [4], and random mutagenesis [5], [6] are all used to stabilize proteins by mutagenesis, but problems still exist, especially in finding simple and general techniques.…”