Thermostabilization of Escherichia coli ribonuclease HI by replacing left-handed helical Lys95 with Gly or Asn.

From the systematic replacements of amino acid residues of Escherichia coli ribonuclease HI with those of its thermophilic counterpart, the basic protrusion domain including region 6 (R6) from residues 91 to 95 was found to increase the structural stability of the mutant protein (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., and Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Further mutagenesis concentrating in the R6 region has revealed that replacements of Lys95 at the left-handed structure with Gly or Asn essentially enhances the protein stability. Gly and Asn substitutions stabilize the protein up to 1.9 kcal/mol and 0.9 kcal/mol in the free energy changes of unfolding, respectively. We propose that the amino acid substitution of left-handed non-Gly residue with Gly or Asn residue can be used as one of the general strategies to enhance protein stability, when such a non-Gly residue itself does not seriously contribute to protein stability.

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The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) KO0552 and X60507.
$ Present address: Basic Research Laboratories, Toray Industries, Inc., 1111, Tebiro, Kamakura, Kanagawa 248, Japan.
5 To whom correspondence and reprint requests should be addressed.
The abbreviations used are: RNase H, ribonuclease H; Re, region 6 of E. coli RNase HI (residues 91-95) defined in Ref. 8. crystal structure of E. coli RNase HI has already been solved (5-7); and 3) the amino acid sequence of T. thermophilus RNase H has been revealed to have 52% identity with the sequence of E. coli RNase HI (4).
The amino acid residues of E. coli RNase HI were systematically replaced with those of its thermophilic counterpart by Kimura et al. (8). It has been found that one of the origins of the structural stability of T. thermophilus RNase H is the amino acid sequence of the basic protrusion domain including region 6 (Rs, residues 91-95) as shown in Fig. 1 (8

MATERIALS AND METHODS
Mutant structural genes were constructed by site-directed mutagenesis using polymerase chain reaction as described previously (8).
For the construction of Rgl-, Eg4-, Gg5-, and Ng5-RNase H, the AAA, GAC, AAA, and AAA codons of Lysgl, Aspg4, L y P , and Lysgs were replaced by CGC, GAA, GGC, and AAC, respectively. Ag5-RNase H was previously constructed (9). In addition, the ACC codon of Thrg2 was silently replaced by ACT to introduce a unique PstI site within the rnhA gene. Construction of the expression plasmids and overproduction and purification of the mutant proteins were also carried out as described previously (8).
Each mutation was confirmed by peptide mapping using Achrornobacter protease I and chymotrypsin, followed by amino acid sequence analysis as described previously (10). Protein concentration was determined from UV absorption at 280 nm. The absorption coefficient A:&' = 2.02 was used assuming that each mutant protein has the same absorption coefficient as that of wild-type E. coli RNase HI (10).
RNase H activity was determined by measurement of the radioactivity of the acid-soluble digestion product from the substrate, 3H-labeled M13 DNA/RNA hybrid, as described previously (11). One unit of enzymatic activity is defined as the amount of enzyme producing 1 pmol of acid-soluble material/min at 37 "C. The specific activity is defined as units of enzymatic activity/mg of protein. CD spectra were measured in 10 mM sodium acetate (pH 5.5) containing 0.1 M sodium chloride at 25 "C on a JASCO 5-600 spectropolarimeter.
Reversible thermal denaturation curves were determined as previously described ( E ) , by monitoring the CD value at 220 nm as temperature increased by 0.7 "C per min. The buffer conditions were 10 mM glycine-HC1 buffer (pH 3.0) containing 1 mM dithiothreitol or 20 mM sodium acetate buffer (pH 5.5) containing both 1 M guanidine hydrochloride and 1 mM dithiothreitol. These conditions were chosen for complete reversibility of the thermal denaturation (11, 12).

RESULTS AND DISCUSSION
The enzymatic activities of mutants E. coli RNase HI varied from 90 to 140% of that of the wild-type E. coli RNase HI, as shown in Table I. They were almost indistinguishable within 67% confidence limits (+30% of the mean of measurement). It means that no current mutations affect the enzymatic activity seriously. The CD spectra of all mutant proteins were almost identical to that of the wild-type E. coli RNase HI, suggesting that the whole protein structures of mutant proteins remain the same as the wild-type protein. Since the R6 region is a separated loop apart from the active site as shown

Thermostabilization by
Left-handed Gly in E. coli RNase HI TABLE I The enzymatic activities and parameters characterizing the thermal denaturation of wild-type and mutant E. coli RNase HI The hydrolysis of the M13 DNA/RNA hybrid with the wild-type and mutant E. coli RNase HI proteins was carried out at 37 "C for 15 min in 10 mM Tris.HCI (pH 8.0) containing 10 mM MgC12, 50 mM NaC1, 1 mM 2-mercaptoethanol, and 10 pg/ml bovine serum albumin. Errors which represent the 67% confidence limits are within 30%. Relative activity represents the specific activity of each mutant protein relative to that of the wild-tvDe Drotein.

Protein
Relative activity The melting temperature, T,,,, is the midpoint of the thermal denaturation curve.
AH,,, is enthalpy change of unfolding, which was derived from van't Hoff analysis. The entropy changes of wild-type protein at T,, AS,, were 0.304 kcal/(mol.K) and 0.275 kcal/(mol.K), at pH 3.0 and 5.5, respectively.
The difference between the free energy change of unfolding of the mutant protein and that of the wild-type protein at T,, AAG" was estimated by the relationship given by Becktel and Shellman (13), AAG, = AT,.AS,. Errors, which represent the 67% confidence limits for the wild-type protein, are f0.4 "C in T,,,, k11.4 kcal/mol in AH,,,, f0.03 kcal/(mol.K) in AS, at pH 3.0, and k0.3 "C in T,, k11.3 kcal/mol in AH,,,, and 0.03 kcal/(mol. K) in AS,,, at pH 5.5, respectively. These were determined from four independent experiments. * AT, is the difference in the melting temperature between the wild-type and mutant proteins.  HI ( 5 , 7). The thick black lines are the region (residues 91-95) including the left-handed structure of Lysg5. Bold circles indicate the active site residues, Asp", G~u~~, and Asp7'. The positions of three amino acid substitutions between the E. coli RNase HI and T. thermophilus RNase H, Lysgl + Arg, Aspg4 -+ Glu, and Lysg5 "+ Gly, are shown with arrows.
in Fig. 1, the mutant proteins can retain similar activities to the wild-type protein as far as the whole protein structures are kept.  Table I. The single mutant protein, Gg5-RNase H, was stabilized by 6.8 "C in T, as compared to the wild-type protein at pH 5.5.
In contrast, the stability of Rgl-RNase H was almost identical to that of the wild-type protein, and Eg4-RNase H was slightly destabilized by 1.6 "C in T, at pH 5.5. The double mutant proteins Rg'/Ggs-RNase H and Eg4/Gg5-RNase H, which contain amino acid substitutions Lysgl + Arg and Aspg4 + Glu in addition to LysS5 + Gly, respectively, have the enhanced thermostabilities similar to that of the triple mutant protein named as %-RNase H (8). However, the double mutant protein Rg1/EQ4-RNase H with the amino acid substitutions of both Lysgl 4 Arg and Aspg4 + Glu, decreased thermostability.
These results indicate that among the three amino acid substitutions at the positions of 91, 94, and 95, Lysg5 + Gly is the determinant of high thermostability of the mutant protein R6-RNase H, which has the same basic protrusion domain as that of T. thermophilus RNase H. In E. coli RNase HI, Lysg5 has the left-handed a-helical structure forming the typical @-hairpin composed of a type-I @-turn followed by a @-bulge (7) (named as 3:5-type; see Ref. 14). Since the region in E. coli RNase HI protrudes into the solution without any intra-molecular interaction with the other part of the protein, it was considered that this region of each mutant protein with Glyg5 has a left-handed structure similar to that Temperature ("C) Only for those proteins for which the crystal structures are available. The numbers on the aligned residues refer to these proteins.
* Characters in parenthei?s indicate that the source organisms are mesophilic (M), moderate thermophilic (T), or extreme thermophilic Successive 5 amino acid residues indicated by one-letter codes, the center of which has the left-handed conformation in the X-ray The backbone dihedral angles at the center of the 5th residue indicated at the Sequence column.
(E) species. structure (indicated by 1). These sequences are aligned following references (21,23,25,27 pendently affects to that of the whole protein ( 8 ) , one of the origins of the high stability of T. thermophilus RNase H can be that the left-handed conformation is occupied by Gly. The left-handed conformations are often observed in native proteins at the @-hairpin loops and at the C termini of ahelices and @-strands (15). This left-handed conformation is not a repetitive secondary structure of native proteins such as a-helix or @-strand but is recognized as one of the important local constituents. Its inverse chirality makes the usual righthanded secondary structures stop, mostly conforming loops at the protein surface. In the common P-hairpin loops, lefthanded a-helical structure and 310-helix structures are observed very frequently, and Gly or Asn residues take the lefthanded conformations much more often than other amino acids (15-17). The left-handed structure of any non-Gly residue has a higher conformation energy (0.5-2 kcal/mol) than the right-handed structure (18-20). While for the symmetric Gly residues, these energies are the same.
Since Asn is also allowed to have the left-handed confor-mation next to Gly (15-17), it was expected that amino acid substitution Lysg5 + Asn can stabilize E. coli RNase HI, too.
As expected, the thermostability of the single mutant protein Ng5-RNase H was increased by 2.9 and 3.2 "C in T,,, at pH 3.0 and 5.5, respectively. On the contrary, the thermostability of Ag5-RNase H was almost identical to that of the wild-type protein, as shown in Fig. 2 and Table I. The role of the short side chain of Asn in stabilizing the left-handed structure can be its ability t o form hydrogen bonding (15). Nicholson et al. (16) reported results different from our current study. They replaced two left-handed residues in phage T4 lysozyme with Gly, and their mutant proteins showed essentially identical stability to the wild-type protein.
In their mutant proteins, some destabilization might occur by losing the intra-molecular hydrogen bonds existing in the wild-type protein (16).
From the present results, the preferences of Gly and Asn for the left-handed conformation can be understood as one of the mechanisms stabilizing protein native conformations. As