Overproduction and preliminary crystallographic study of ribonuclease H from Escherichia coli

To test whether the combination of multiple thermostabilizing mutations is a useful strategy to generate a hyperstable mutant protein, five mutations, Gly23-->Ala, His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His or Asn, were simultaneously introduced into Escherichia coli ribonuclease HI. The enzymatic activities of the resultant quintuple mutant proteins, 5H- and 5N-RNases HI, which have His and Asn at position 134, respectively, were 35 and 55% of that of the wild-type protein. The far-UV and near-UV CD spectra of these mutant proteins were similar to those of the wild-type protein, suggesting that the mutations did not seriously affect the tertiary structure of the protein. The differences in the free energy change of unfolding between the wild-type and mutant proteins, delta delta G, were estimated by analyzing the thermal denaturation of the proteins by CD. The 5H-RNase HI protein, which was slightly more stable than the 5N-RNase HI, was more stable than the wild-type protein by 20.2 degrees C in Tm and 5.6 kcal/mol in delta G at pH 5.5. In addition, the 5H-RNase HI was highly resistant to proteolysis and acid denaturation. The effects of each mutation on the thermal stability and the susceptibility to chymotryptic digestion were nearly cumulative, and the 5H-RNase HI undergoes chymotryptic digestion at a rate that is 41 times slower than that of the wild-type protein. Good correlation was observed between the thermal stability and the resistance to chymotryptic digestion for all proteins examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

High Resistance of Escherichia coli Ribonuclease HI Variant with Quintuple Thermostabilizing Mutations to Thermal Denaturation, Acid Denaturation, and Proteolytic Degradation

Akasako¹,

Haruki²,

Oobatake³

et al. 1995

“…Protein Preparation. E. coli N4830-1 with plasmid vector pPL801 controlled by the bacteriophage X PL promoter was utilized for the overproduction of RNase H (Kanaya et al, 1989). At 42 °C, production of the protein was induced after multiplication at 30 °C.…”

Section: Methodsmentioning

confidence: 99%

Assignments of backbone proton, carbon-13, and nitrogen-15 resonances and secondary structure of ribonuclease H from Escherichia coli by heteronuclear three-dimensional NMR spectroscopy

Yamazaki¹,

Yoshida²,

Kanaya³

et al. 1991

The assignments of individual magnetic resonances of backbone nuclei of a larger protein, ribonuclease H from Escherichia coli, which consists of 155 amino acid residues and has a molecular mass of 17.6 kDa are presented. To remove the problem of degenerate chemical shifts, which is inevitable in proteins of this size, three-dimensional NMR was applied. The strategy for the sequential assignment was, first, resonance peaks of amides were classified into 15 amino acid types by 1H-15N HMQC experiments with samples in which specific amino acids were labeled with 15N. Second, the amide 1H-15N peaks were connected along the amino acid sequence by tracing intraresidue and sequential NOE cross peaks. In order to obtain unambiguous NOE connectivities, four types of heteronuclear 3D NMR techniques, 1H-15N-1H 3D NOESY-HMQC, 1H-15N-1H 3D TOCSY-HMQC, 13C-1H-1H 3D HMQC-NOESY, and 13C-1H-1H 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with 15N or with 13C. This method gave a systematic way to assign backbone nuclei (N, NH, C alpha H, and C alpha) of larger proteins. Results of the sequential assignments and identification of secondary structure elements that were revealed by NOE cross peaks among backbone protons are reported.

“…Among the various RNase H proteins, E. coli RNase HI has been most extensively studied for its structure-function relationship by various techniques, including X-ray analysis (Katayanagi etal., 1990(Katayanagi etal., ,1992Yang et al, 1990), site-directed et al, 1990; Yamazaki et al, 1991Yamazaki et al, , 1993Nakamura et al, 1991;Oda etal., 1991Oda etal., ,1992Oda etal., ,1993). The enzyme is composed of a single polypeptide chain of 155 amino acid residues (Kanaya & Crouch, 1983) with an isoelectric point (p/) of 9.0 (Kanaya et al, 1989). On the basis of a series of investigations, three amino acid residues with carboxyl groups (Asp 10, Glu48, and Asp70) are postulated to form the catalytic site of the enzyme.…”

mentioning

confidence: 99%

Individual Ionization Constants of All the Carboxyl Groups in Ribonuclease HI from Escherichia coli Determined by NMR

Oda

Yamazaki²,

Nagayama³

et al. 1994

147

170

All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme that cleaves the RNA strand of a RNA/DNA hybrid, were pH-titrated, and their ionization constants (pKa) were determined from an analysis of the pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1H-13C heteronuclear two-dimensional NMR. The pKa values in the enzyme varied widely among individual residues, for example, in the unusual pKa values for two important catalytic residues, Asp10 (pKa 6.1) and Asp70 (pKa 2.6). Moreover, remarkable two-step titrations were observed for these carboxylates. The binding of Mg2+ ion to the enzyme, which is the cofactor necessary for catalytic activity, caused no significant change in the pKa values of the carboxyl groups, except for that of Asp10. The variations of the pKas that were dependent on the microenvironment in the protein were theoretically reproduced to compare with the experimental results by a numerical calculation, using a continuum electrostatic model. Most of the significant pKa decreases were brought about through strong electrostatic interactions with the neighboring basic amino acids, Arg or Lys. The pKa shifts and the two-step titrations of Asp10 and -70, which are close to each other, were interpreted to be due to the neighboring effect of two functional groups, as observed in the interacting titratable groups of a dicarboxyl compound or in the active site carboxylates of lysozyme and aspartic protease. The role of Asp10 in the catalytic action is either to be the proton donor to the RNA moiety or the binding partner of the Mg2+ ion cofactor. Asp70, on the other hand, is considered to be the proton acceptor from a water molecule.