X‐ray crystallographic and calorimetric studies of the effects of the mutation Trp59→ Tyr in ribonuclease T1

Abstract

Two mutants of ribonuclease T1 (RNaseTl), [59-tyrosine]ribonuclease T1 (W59Y) and [45-tryptophan,59-tyrosine]ribonuclease T1 (Y45W/W59Y) possess between 150% and 190% wild-type activity. They have been crystallised as complexes of the inhibitor 2'-guanylic acid and analysed by X-ray diffraction at resolutions of 0.23 nm and 0.24 nm, respectively. The space group for both is monoclinic, P2,, with two molecules/asymmetric unit, W59Y: a Ribonuclease T1 (RNaseTl) isolated from Aspergillus oryzae is a small endonuclease consisting of 104 amino acids. It cleaves single-stranded RNA specifically after guanosine. Two isoforms with either Gln or Lys at position 25 have been identified, both having comparable activity towards RNA. Due to its small size and inherent stability, RNaseTl is used as a model system in protein stability and folding studies, and consequently much biochemical data for this protein have been accumulated (Heinemann and Hahn, 1989;Pace et al. 1991). Following the cloning of its gene in Escherichia coli, mutants of RNaseTl produced by sitedirected mutagenesis have become available (Quaas et al., 1988a, b).The sequence alignment of several microbial enzymes of the RNaseTl family (Heinemann and Hahn, 1989) indicated that the only tryptophan in RNaseTl, Trp59, was conservatively substituted by the aromatic amino acids phenylalanine or tyrosine. This implied that Trp59 could possibly be replaced by tyrosine in RNaseTl without impairing the enzyme activity significantly. The replacement of Trp59 by tyrosine was studied by enzyme kinetics and in three additional mu-

Analysis of a Water Mediated Protein−Protein Interactions within RNase T1,

“…A ratio of the van't Hoff enthalpy ∆H vH to the calorimetric enthalpy ∆H trs of one is a necessary criterion for a two-state transition. For all our measurements, this ratio was close to one (data not shown), which is in good agreement with the results of other authors (31,34,35). According to the reversible two-state equilibration, DSC can be used to measure the thermodynamic parameters of unfolding of these proteins.…”

Impact of Point Mutations on the Structure and Thermal Stability of Ribonuclease T1 in Aqueous Solution Probed by Fourier Transform Infrared Spectroscopy

“…It is well-known that proteins in D20 solutions show higher Tm values compared to those found in H2O solutions (Talluri & Scheraga, 1990; Yamamoto & Tasumi, 1991). More importantly, however, the relative differences between the Tm values for the wildtype RNase Tl and the three mutants determined by infrared spectroscopy in D20-buffer are in good agreement with the corresponding differences between the Tm values derived from scanning calorimetric data obtained in H20-buffer (Schubert et al, 1994). (iii) While the spectrum of the wild-type protein at 70 °C is practically identical with that of the mutant Y45 W/ W59Y, their spectra at 21 °C show minor differences, particularly with respect to the position of the tyrosine band (see Figure 4B).…”

Calorimetric Investigation of Thermal Stability and Ligand‐binding Characteristics of Disulfide‐bond‐cleaved Ribonuclease T1

“…Recently, thermodynamic stability data about wild-type ribonuclease T, have been reported (Barone et al, 1992;Schubert et al, 1994;Yu et al, 1994;Plaza del Pino et al, 1992). Unfortunately, only a qualitative comparison with the data presented in this study is possible because of the necessity of choosing a different solution system (2 M NaCl), which increases the thermodynamic parameters t, and A G significantly.…”