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Ribonuclease T1 is an enzyme that cleaves single-stranded RNA with high specificity after guanylyl residues. Although this enzyme is a very good characterized protein with respect to structure and enzymatic function, we were only recently successful in generating RNase T1-RV, a variant where the specificity was changed from guanine to purine. As this change of substrate specificity was made at the cost of activity, the aim was now to further improve the overall activity of the enzyme. Therefore, we have substituted the tryptophan in position 59 by tyrosine. This substitution led to an increase of enzymatic activity in comparison to variant RV to 425%. As the extent of this enhancement is unique so far we have crystallized and analyzed the structure of this variant in order to get more insights into the reasons for this. Here, we present the crystal structure of this so-called RNase T1-R2 at 2.1A resolution. The structure was determined by molecular replacement using the coordinates of the RV variant (PDB entry: 1Q9E). The data were refined to an R-factor of 18.7% and R(free) of 24%, respectively. The asymmetric unit contains three molecules and the crystal packing is very similar to that of variant RV.

Attempts to alter the guanine specificity of ribonuclease T1 (RNase T1) by rational or random mutagenesis have failed so far. The RNase T1 variant RV (Lys41Glu, Tyr42Phe, Asn43Arg, Tyr45Trp, and Glu46Asn) designed by combination of a random and a rational mutagenesis approach, however, exhibits a stronger preference toward adenosine residues than wild-type RNase T1. Steady state kinetics of the cleavage reaction of the two dinucleoside phosphate substrates adenylyl-3',5'-cytidine and guanylyl-3',5'-cytidine revealed that the ApC/GpC ratio of the specificity coefficient (k(cat)/K(m)) was increased approximately 7250-fold compared to that of the wild-type. The crystal structure of the nucleotide-free RV variant has been refined in space group P6(1) to a crystallographic R-factor of 19.9% at 1.7 A resolution. The primary recognition site of the RV variant adopts a similar conformation as already known from crystal structures of RNase T1 not complexed to any nucleotide. Noteworthy is a high flexibility of Trp45 and Asn46 within the three individual molecules in the asymmetric unit. In addition to the kinetic studies, these data indicate the participation of Asn46 in the specific recognition of the base and therefore a specific binding of adenosine.

Although ribonuclease T1 (RNase T1) is one of the best-characterized proteins with respect to structure and enzymatic action, numerous attempts at altering the specificity of the enzyme to cleave single-stranded RNA at the 3'-side of adenylic instead of guanylic residues by rational approaches have failed so far. Recently we generated and characterized the RNase T1 variant RV with a 7200-fold increase in adenylyl-3',5'-cytidine (ApC)/guanylyl-3',5'-cytidine (GpC) preference, with the guanine-binding loop changed from 41-KYNNYE-46 (wt) to 41-EFRNWN-46. Now we have introduced the asparagine residue at position 46 of the wild-type enzyme as a single-point mutation in variant E46N and in combination with the Y45W exchange also occurring in RV. Both variants show an improved ApC/GpC preference with a 1450-fold increase for E46N and a 2100-fold increase for Y45W/E46N in comparison to wild-type activity. We also addressed the challenge of altering enzyme specificity with an evolutionary approach. We have randomly introduced point mutations into the RNase T1 wild-type gene and into the gene of the variant RV with different mutation rates. Altogether we have screened about 100,000 individual clones for activity on RNase indicator plates; 533 of these clones were active. A significant change in substrate specificity towards an ApC preference could not be observed for any of these active variants; this demonstrated the magnitude of the challenge to alter the specificity of this evolutionary perfected enzyme.

Ribonuclease T1 was biosynthesized, with all four prolines (13)C-labeled in the peptide C[double bond]O bond, using a proline auxotrophic yeast strain of Saccharomyces cerevisiae. The (13)C- and (12)C-proline isotopomers of ribonuclease T1 were investigated by infrared spectroscopy in the thermally unfolded and natively folded state at 80 and 20 degrees C, respectively. In the thermally unfolded state, both proteins established almost indistinguishable spectral features in the secondary structure sensitive amide I region. In contrast, the spectra measured at 20 degrees C revealed substantial qualitative and quantitative differences, though parallel analysis by circular dichroism suggested identical native folds for both isotopomers. Major spectral differences in the infrared spectra were detected at 1626 and 1679 cm(-1), which are diagnostic marker bands for antiparallel beta-sheets in ribonuclease T1 and at 1645 cm(-1), a region that is characteristic for the infrared absorption of irregular structures. Starting with the known three-dimensional structure of ribonuclease T1, the observed effects of the isotope labeling are discussed on the basis of transition dipole coupling between the (12)C[double bond]O and (13)C[double bond]O groups. The experimental results were confirmed by transition dipole coupling calculations of the amide I manifold of the labeled and unlabeled variant.

An RNA sequence showing high stability with respect to digestion by ribonuclease T1 (RNase T1) was isolated by in vitro selection from an RNA library. Although ribonuclease T1 cleaves single-stranded RNA specifically after guanosine residues, secondary structure calculations predict several guanosines in single-stranded areas. Two of these guanosines are part of a GGCA-tetraloop, a recurring structure element in the secondary structure predictions. Molecular dynamics simulations of the conformation space of the nucleotides involved in this tetraloop show on the one hand that the nucleic acid backbone of the guanosines cannot realise the conformation required for cleavage by RNase T1. On the other hand, it could be shown that an RNA molecule not forced into a tetraloop occupies this conformation several times in the course of the simulation. The simulations confirm the GGCA-tetraloop as an RNase-stable secondary structure element. Our results show that, besides the known prerequisite of a single-stranded RNA, RNase T1 has additional demands on the substrate conformation.

RNA-oligonucleotides with defined single-stranded stretches were designed to investigate the minimal requirements of a ribonuclease T1 substrate. It could be shown, that RNase T1 cleaves single-stranded RNA after a unique guanosine flanked by two double-stranded areas. However, the turnover of such a G-gap is significantly lower than that of a gap of two, three or four nucleotides.

We report a new method for studying the activity of hydrolytic enzymes. Fluorescence correlation spectroscopy was used to observe online the hydrolyzation of a rhodamine B-labeled substrate by ribonuclease T1. A gapped heteroduplex substrate - a hybrid of a ribooligonucleotide and two smaller complementary deoxyribooligonucleotides - was immobilized via biotin to a streptavidin-coated surface of a coverslip. The reported method opens the possibility to study the cleavage of small substrates differing only slightly in molecular weight from the enzyme reaction product. The use of fluorescence correlation spectroscopy allows the detection of very low enzyme concentrations (down to 10(-21) mol 0.05 fM of RNase T1, corresponding to about 600 RNase T1 molecules in 0.02 ml).

The ribonuclease T1 variant 9/5 with a guanine recognition segment, altered from the wild-type amino acid sequence 41-KYNNYE-46 to 41-EFRNWQ-46, has been cocrystallised with the specific inhibitor 2'-GMP. The crystal structure has been refined to a crystallographic R factor of 0.198 at 2.3 A resolution. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 A, 2'-GMP is fixed to the primary recognition site due to increased aromatic stacking interactions. The phosphate group of 2'-GMP is located about 4.2 A apart from its position in wild-type ribonuclease T1-2'-GMP complexes, allowing a Ca(2+), coordinating this phosphate group, to enter the binding pocket. The crystallographic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine affinity and turnover rate. The presence of Ca(2+) was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent., (Copyright 1999 Academic Press.)

Attempts to modify the guanine specificity of ribonuclease T1 (RNase T1) by rationally designed amino acid substitutions failed so far. Therefore, we applied a semirational approach by randomizing the guanine binding site. A combinatorial library of approximately 1.6 million RNase T1 variants containing permutations of 6 amino acid positions within the recognition loop was screened on RNase indicator plates. The specificity profiles of 180 individual clones showing RNase activity revealed that variant K41S/N43W/N44H/Y45A/E46D (RNaseT1-8/3) exhibits an altered preference toward purine nucleotides. The ApC/GpC preference in the cleavage reaction of this variant was increased 4000-fold compared to wild-type. Synthesis experiments of dinucleoside monophosphates from cytidine and the corresponding 2'3'-cyclic diesters using the reverse reaction of the transesterification step showed a 7-fold higher ApC synthesis rate of RNase 8/3 than wild-type, whereas the GpC synthesis rates for both enzymes were comparable. This study shows that site-directed random mutagenesis is a powerful additional tool in protein design in order to achieve new enzymatic specificities.

Small-angle X-ray scattering of RNase T1 with intact disulfide bonds was measured at 20 degrees and 60 degrees C in order to get insight into the structural changes of the protein caused by thermal denaturation. The radius of gyration increases from R(G)= 1.43 nm to R(G) = 2.21 nm. The conformations of the molecules at 60 degrees C are similar to those of ring-shaped random walk chains. However, the molecules are more compact than one would expect under theta conditions due to attractive interactions between the chain segments. The volume needed for free rotation of the thermally unfolded protein molecules about any axis in solution is five times greater than in the native state whereas the hydrodynamic effective volume is increasing only two times.

The enzymatic activity of many ribonucleases (RNases) depends on two histidines, as in RNase A, or one histidine and/or glutamate, as in microbial RNases belonging to the T1 family. In RNase T1, substitution of either one or both of the two histidines at positions 40 and 92 by a variety of other amino acids reduces the activity more than 100-fold. However, the double variant His40-->Asp/His92-->Asp retains a significant residual enzymatic activity towards RNA and guanylyl-3',5'-cytidine, indicating that a pair of substituted side chains in these positions, both with acid functionality, can confer enzymatic activity. It was shown that the substitution of histidine with glutamate in the variant His40-->Glu yields an enzyme with drastically reduced activity and leads to inactivation in the His92-->Glu, His40-->Glu/His92-->Glu variants. For the variants where histidine is substituted with aspartate we found measurable activity from 1% (His40-->Asp) to 6% (His40-->Glu/His92-->Asp) towards RNA.

Ribonuclease T1 can be unfolded and refolded without forming noticeable amounts of aggregates allowing to characterise the dimensions of a protein in different denatured states in terms of the Stokes radius RS. Upon thermal unfolding RS increases from 1.74 nm at 20 degrees C to 2.14 nm at 60 degrees C. By contrast, RS = 2.40 nm was obtained at 5.3 M guanidinium chloride (GuHCl) and 20 degrees C. Heating from 20 degrees C to 70 degrees C in the presence of 5.3 M GuHCl led to a 5% decrease in RS.

We have studied ribonuclease T1 (EC 3.1-27.3)-catalysed synthesis of guanylyl-(3'-->5')cytidine in frozen aqueous reaction mixtures at -10 degrees C and in solution at 0 degree C in order to investigate whether ribonuclease-catalysed synthetic reactions can take advantage of the yield-increasing effect of freezing as was reported for protease-catalysed peptide synthesis. Under frozen state conditions, substantially increased yields of GpC were obtained compared to the reactions in solution. From the fact that no irreversible hydrolysis of the 2'3'-cyclic donor was observed it can be concluded that transesterification of the newly formed phosphodiester bond is the most important yield-limiting factor in ribonuclease T1-catalysed dinucleoside phosphate synthesis.

Protein folding is often retarded by the cis reversible trans isomerizations of prolyl peptide bonds both in vitro and in vivo. An important role for the folding mechanism is well established for the prolyl peptide bonds that are cis in the native protein, but not for those that are trans. Here we investigated the role of trans-Pro73 for the folding of ribonuclease T1 (which additionally contains two cis-prolines) by comparing the wild-type protein with the Pro73-->Val variant. The Pro-->Val substitution led to a destabilization of the folded protein by 8.5 kJ/mol, which is explained by the strong, 25-fold increase in the rate of unfolding. In contrast, the rates and amplitudes of the fast and slow refolding reactions were virtually unchanged. trans-Proline residues remain largely trans after unfolding, and therefore their contributions to the observed folding kinetics should indeed be insignificant for proteins which also contain one or more cis prolines. The cis-proline residues dominate the kinetics of refolding, because almost all slow-folding molecules contain the respective incorrect (trans) isomers, and because trans-->cis isomerizations are slower than cis-->trans isomerizations. The inability to detect contributions from a trans-proline to the kinetics of folding does not imply that this proline is non-essential for folding in the sense that its cis reversible trans isomerization is energetically uncoupled from conformational folding.

Electrostatic interactions between charged residues and the helix dipole in a protein were investigated by protein engineering methods. In ribonuclease T1, two surface-exposed acidic residues (Glu28 and Asp29) are located near the carboxyl terminus of the alpha-helix between residues 13 and 29. They were replaced, individually and in concert, by the uncharged amides Gln28 and Asn29, and the stabilities of the wild-type protein and its variants were determined as a function of pH. The effects of the two mutations are additive. Either one leads to a marginal destabilization by 0.7 kJ/mol at pH 2 but to a strong stabilization by about 3.2 kJ/mol at pH 7. This suggests that the deprotonations of Glu28 and Asp29 reduce the free energy of stabilization of folded ribonuclease T1 by about 4 kJ/mol each. This destabilization is probably caused by unfavorable electrostatic interactions of Glu28 and Asp29 with the negative end of the helix dipole. The activation energies for the unfolding of the different variants of ribonuclease T1 change in parallel with the differences in the thermodynamic stability when the pH is varied. This indicates that the unfavorable electrostatic interactions of Glu28 and Asp29 are lost very early in unfolding, and are not present in the activated state of unfolding.

We undertook a detailed comparative analysis of the infrared spectra of wild-type ribonuclease T1 and three mutants: two single mutants, Tyr-45-->Trp (Y45W) and Trp-59-->Tyr (W59Y), and a double mutant, Tyr-45-->Trp/Trp-59-->Tyr (Y45W/W59Y). These mutants were selected because they are known to affect the activity of the enzyme. The structural differences were evaluated by using peptide backbone and side-chain "marker" bands as conformation-sensitive monitors. All mutations lead to a decrease of the thermal transition temperature, though the mutation Tyr-45-->Trp affects the Tm to a lesser degree than the replacement of Trp-59 by Tyr, both in the single (W59Y) and in the double (Y45W/W59Y) mutant. Small changes in the protein backbone conformation and in the microenvironment of certain amino acids, induced by the point mutations, could be detected. In particular, we found subtle differences in the hydrogen bonding pattern of the beta-strands in the mutants W59Y and Y45W/W59Y, compared to that in wild-type RNase T1 and in the mutant Y45W. Practically identical spectra in the amide I region were obtained for the double mutant Y45W/W59Y and the single mutant W59Y, demonstrating that it is the change from Trp to Tyr in position 59 (located at the interface between the alpha-helix and a beta-strand) which affects the overall protein conformation. The mutation Tyr to Trp in position 45, on the other hand, has practically no impact on the polypeptide backbone conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Two mutants of ribonuclease T1 (RNaseT1), [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(1), with two molecules/asymmetric unit, W59Y: a = 4.934 nm, b = 4.820 nm, c = 4.025 nm, beta = 90.29 degrees. Y45W/W59Y: a = 4.915 nm, b = 4.815 nm, c = 4.015 nm, beta = 90.35 degrees. Compared to wild-type RNaseT1 in complex with 2'-guanylic acid (2'GMP) both mutant inhibitor complexes indicate that the replacement of Trp59 by Tyr leads to a 0.04-nm inward shift of the single alpha-helix and to significant differences in the active-site geometry, inhibitor conformation and inhibitor binding. Calorimetric studies of a range of mutants [24-tryptophan]ribonuclease T1 (Y24W), [42-tryptophan]ribonuclease T1 (Y42W), [45-tryptophan]ribonuclease T1 (Y45W), [92-alanine]ribonuclease T1 (H92A) and [92-threonine]ribonuclease T1 (H92T) with and without the further mutation Trp59-->Tyr showed that mutant proteins for which Trp59 is replaced by Tyr exhibit slightly decreased thermal stability.

Recombinant ribonuclease (RNase) T1 variants were characterized kinetically taking into account the different reactions catalyzed by this enzyme. In addition to established assays, monitoring the transesterification activity, a photometric assay for fast screening of RNase T1 and variants thereof for ester hydrolysis activity is described, which is based on the application of phenol red as pH indicator. Moreover we established an HPLC assay to evaluate RNase T1 variants by their ability to carry out the transesterification towards an internucleotide diphosphoester (reverse or synthetic activity). In this way we found that the transesterification and hydrolyzing activities of variants change in various directions though in all reactions the same active site and the same transition state are involved. The variant where Tyr42 has been replaced by Trp performs RNA synthesis better than the wild type protein. The scheme of the hypothetic RNase T1 mechanism had to be improved to take into account the non processive character of the reaction.

The crystal structure of the complex between ribonuclease T1 and 3'GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 N epsilon H+ activates O2'-H, (d) Glu58 carboxylate acts as base and His92 N epsilon H+ as acid in a general acid-base catalysis. The crystals have the monoclinic space group P2(1), a = 4.968 nm, b = 4.833 nm, c = 4.048 nm, beta = 90.62 degrees with two molecules in the asymmetric unit. The structure was determined by molecular replacement and refined to R = 15.3% with 11,338 data > or = 1 sigma (Fo) in the resolution range 1.0-0.2 nm; this includes 180 water molecules and two Ca2+. The structure of ribonuclease T1 is as previously observed. 3'GMP is bound in syn conformation; guanine is located in the specific recognition site, the ribose adopts C4'-exo puckering, the ribose phosphate is extended with torsion angle epsilon in trans. The O2'-H group is activated by accepting and donating hydrogen bonds from His40 N epsilon H+ and to Glu58 O epsilon 1; the phosphate is hydrogen bonded to Glu58 O epsilon 2H, Arg77 N epsilon H+ and N eta 2H+, Tyr38 O eta H, His92 N eta H+. The conformation of ribose phosphate is such that O2' is at a distance of 0.31 nm from phosphorus, and opposite the P-OP3 bond which accepts a hydrogen bond from His92 N epsilon H+; we infer from a model building study that this bond is equivalent to the scissile P-O5' in a substrate GpN.

Using point mutated overproducing strains of E. coli, ribonuclease T1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp, Tyr45Trp or Trp59Tyr and the corresponding double substitutions Tyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr. Steady state kinetics of the transesterification reaction for the two dinucleoside monophosphate substrates guanylyl-3',5'-cytidine and guanylyl-3',5'-adenosine indicate that the tryptophan can be introduced in different positions within the ribonuclease T1 molecule without abolishing enzymatic activity. The Trp59Tyr exchange even enhances catalysis of the cleavage reaction (kcat/Km) relative to the wild type enzyme and similar effects are found with single tyrosine to tryptophan substitutions. For the pH dependencies of the guanylyl-3',5'-cytidine transesterification reaction of wild type ribonuclease T1 and of the variants, typically bell-shaped curves are observed with a plateau in the range pH 4.5-7.0. Their shapes and slopes indicate that the enzymes are comparable in their macroscopic pKa values. At pH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-fold higher transesterification activity for guanylyl-3',5'-adenosine and a 2-fold increase for guanylyl-3',5'-cytidine compared to the wild type enzyme, i.e. this variant catalyses the transesterification of the substrate guanylyl-3',5'-adenosine with the same or better efficiency as guanylyl-3',5'-cytidine.

The secondary structure of ribonuclease T1 (RNase T1) in aqueous solution and its temperature-induced structural changes have been investigated by Fourier-transform infrared (FT-IR) spectroscopy. 13 to 14% alpha-helix and 32 to 33% beta-sheet were estimated from the resolution-enhanced FT-IR spectra, in agreement with the crystal structure which indicates 16% alpha-helix and 35% beta-sheet. Specific IR-marker bands are assigned to the different beta-sheet structures, to the slightly bent alpha-helix, and to beta-turn and irregular conformations present in RNase T1. The temperature dependence of the infrared spectra shows that the thermal unfolding and refolding of RNase T1 is fully reversible. This permitted the detailed analysis of structural changes that occur as a function of temperature by evaluating quantitatively the various secondary structure-related amide I band components and some amino acid side-chain vibrations as specific monitors. The secondary structure of RNase T1 is essentially retained in the temperature range between 20 and 50 degrees C. Significant perturbation of protein structure is initiated between 50 and 55 degrees C within regions of beta-sheet structures while the alpha-helix remains virtually intact up to 55 degrees C suggesting a "premelting" of RNase T1. Between 55 and 60 degrees C, a highly co-operative unfolding process is indicated by the simultaneous breakdown of all secondary structure components and by distinct changes of some specific side-chain vibrations. An analysis of the amide I band contour of RNase T1 at 70 degrees C proves that the unfolded state is predominantly, but not completely, irregular or "random coil". Residual, turn-like structures persisting even in the unfolded state are suggested by minor, turn related band components in the amide I region. From IR-spectra collected along a linear temperature gradient, intensity/temperature and frequency/temperature profiles were constructed using some peptide backbone and amino acid side-chain marker bands as local, structure-sensitive monitors. From these profiles individual transition temperatures tm and transition enthalpies delta H (van't Hoff) were calculated. The tm and delta H values revealed a small but distinct hysteresis between repetitive cycles of unfolding and refolding of the protein, suggesting slow refolding kinetics of RNase T1. Furthermore, the various infrared "marker bands" indicate a slightly different response towards temperature increase/decrease for different regions of the protein. The data demonstrate that infrared spectroscopy permits both the detailed analysis of structural changes occurring in a protein as a function of temperature and the determination of thermodynamic parameters characterizing its folded/unfolded state transition.

The refolding of ribonuclease T1 involves two major slow processes that exhibit properties of prolyl isomerization reactions. A comparison of the wild-type protein and a designed variant where the cis Ser54-Pro55 bond was replaced by a Gly54-Asn55 bond indicated that the faster of these reactions is the isomerization of Pro55. Here we report the replacement of the other cis proline of ribonuclease T1 at position 39 by alanine. The Pro39Ala variant is similar to the wild-type protein in secondary and tertiary structure, and the enzymatic activity towards RNA and a dinucleotide substrate remains almost unchanged. The fluorescence emission of the single Trp59 is lowered by the Pro39Ala substitution, probably because Trp59 is in close contact to Pro39 in wild-type ribonuclease T1. Unlike the substitution of cis Pro55, the Pro39Ala mutation is strongly destabilizing and reduces the Gibbs free energy of the folded protein by about 20 kJ/mol. Pro39 is buried in native RNase T1 and located near the active site. The observed destabilization could originate from the presence of a cis alanyl bond in the Pro39Ala variant or from a local distortion caused by the incorporation of a trans alanyl peptide bond in the interior of the protein. In the refolding kinetics the replacement of Pro39 leads to a disappearance of the fast-refolding species. Refolding still involves two consecutive slow steps. The first and faster step could be the isomerization of the remaining cis Pro55. The second, very slow step is a novel reaction that appears to have no counterpart in the refolding of the wild-type protein. All mutant molecules must undergo this reaction before reaching the native state. These major changes in the folding kinetics strongly indicate that cis-Pro39 is indeed of major importance for the folding of the wild-type protein. They indicate, moreover, that some new feature of protein folding kinetics is observed in these studies of the Pro39Ala variant.

Purification of recombinant RNase T1 and its mutants has been improved by optimizing bacterial growth conditions, periplasmic fraction preparation and the use of a precolumn. The main part of the chromatographic separation could be automated due to the reproducibility of the procedure.

The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)

The replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline-limited reaction that is unusually slow in the refolding of the wild-type protein. The distribution of fast- and slow-folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp-59 is in close contact to Pro-39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with the trans----cis reisomerization of the Tyr-38-Pro-39 bond at the stage of a native-like folding intermediate. The steric hindrance is abolished either by changing Trp-59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild-type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal that trans----cis isomerization of Pro-39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate rate-limiting steps by trapping partially folded structures.

For 30 years, the prevailing view has been that the hydrophobic effect contributes considerably more than hydrogen bonding to the conformational stability of globular proteins. The results and reasoning presented here suggest that hydrogen bonding and the hydrophobic effect make comparable contributions to the conformational stability of ribonuclease T1 (RNase T1). When RNase T1 folds, 86 intramolecular hydrogen bonds with an average length of 2.95 A are formed. Twelve mutants of RNase T1 [Tyr----Phe (5), Ser----Ala (3), and Asn----Ala (4)] have been prepared that remove 17 of the hydrogen bonds with an average length of 2.93 A. On the basis of urea and thermal unfolding studies of these mutants, the average decrease in conformational stability due to hydrogen bonding is 1.3 kcal/mol per hydrogen bond. This estimate is in good agreement with results from several related systems. Thus, we estimate that hydrogen bonding contributes about 110 kcal/mol to the conformational stability of RNase T1 and that this is comparable to the contribution of the hydrophobic effect. Accepting the idea that intramolecular hydrogen bonds contribute 1.3 +/- 0.6 kcal/mol to the stability of systems in an aqueous environment makes it easier to understand the stability of the "molten globule" states of proteins, and the alpha-helical conformations of small peptides.

Based on the proposal that ribonucleases cleave the RNA phosphodiester bond with a mechanism involving pentacovalent phosphorous as transition state, complexes of guanosine and inosine with vanadate-(IV, V), molybdate-(VI), tungstate-(VI), chromate-(VI) and hexacyanochromate-(III) were synthesized and probed as inhibitors of recombinant ribonuclease T1, obtained from an E. coli. overproducing strain. The apparent dissociation constants of these inhibitors and RNase T1, as determined by Michaelis-Menten kinetics, vary between 0.5-0.9 microM and indicate very strong binding, 100- to 1000-fold stronger than the binding of guanosine (Kd = 545 microM) and inosine (Kd = 780 microM), and 50-100-fold stronger than the binding of the product 3' GMP (Kd = 55 microM). Therefore the synthesized inhibitors may be considered as genuine transition state analogs for the enzyme.

Uniformly 15N-enriched ribonuclease T1 (RNase T1) was obtained from Escherichia coli by recombinant techniques. Heteronuclear 1H, 15N-shift correlation spectra were recorded utilizing proton detection. Direct 1H, 15N connectivities were established applying the heteronuclear multiple-quantum coherence technique. Additional 1H, 1H-TOCSY or 1H, 1H-NOESY transfer steps allowed for sequential assignments. Nitrogen atoms without directly bonded protons were detected by means of the heteronuclear multiple-bond correlation experiment. Signals emerging from 15NH and 15NH2 groups were distinguished by heteronuclear triple-quantum filtering methods. 119 nitrogen resonances out of the expected 127 were assigned unambiguously; in addition, previously obtained proton assignments were extended. Preliminary 1H, 15N NMR investigation were performed on the RNase-T1-3'GMP inhibitor complex. Results were interpreted with respect to nucleotide binding.

Using an Escherichia coli overproducing strain secreting Aspergillus oryzae RNase T1, we have constructed and characterized mutants where amino acid residues in the catalytic center have been substituted. The mutants are His40----Thr, Glu58----Asp, Glu58----Gln, His92----Ala and His92----Phe. His92----Ala and His92----Phe mutants are inactive. On the basis of their kcat/Km values, the mutants Glu58----Asp and Glu58----Gln show 10% and 7% residual activity, relative to wild-type RNase T1, whereas the His40----Thr mutant shows 2% activity. The effect of amino acid substitutions on the enzymatic activity of RNase T1 lends further support for a mechanism where Glu58 (possibly activated by His40 and His92 act as general base and acid respectively; this is discussed in terms of the known three-dimensional structure of the enzyme.

Fluorescence titrations and temperature-jump relaxation experiments were performed as a function of temperature on ribonuclease T1 with the inhibitors 2'GMP and 3'GMP to obtain information on the energetics and molecular events controlling the binding of those inhibitors. Results from the titration and temperature-jump experiments were in agreement concerning the equilibrium constant. The larger equilibrium constant for 2'GMP is enthalpic in origin and is due to both a higher on rate and a lower off rate as compared to 3'GMP. On rates for both inhibitors appear to be below the diffusion controlled limit, apparently due to conformational changes in the portion of the active site responsible for recognition of the guanine base. Comparison of the measured enthalpic and entropic terms associated with the equilibrium constant determined from the fluorescence titrations are in disagreement with those calculated from the on and off rates indicating the presence of an induced conformational change in the 2'GMP-enzyme complex. This second conformational change appears to be due to additional interactions between 2'GMP and the catalytic portion of the active site, which may also be responsible for the differences in the binding constant, the on rate and the off rate between 2'GMP and 3'GMP.

The conformational stability of recombinant Lys25-ribonuclease T1 has been determined by differential scanning microcalorimetry (DSC), UV-monitored thermal denaturation measurements, and isothermal Gdn.HCl unfolding studies. Although rather different extrapolation procedures are involved in calculating the Gibbs free energy of stabilization, there is fair agreement between the delta G degrees values derived from the three different experimental techniques at pH 5, theta = 25 degrees C: DSC, 46.6 +/- 2.1 kJ/mol; UV melting curves, 48.7 +/- 5 kJ/mol; Gdn.HCl transition curves, 40.8 +/- 1.5 kJ/mol. Thermal unfolding of the enzyme is a reversible process, and the ratio of the van't Hoff and calorimetric enthalpy, delta HvH/delta Hcal, is 0.97 +/- 0.06. This result strongly suggests that the unfolding equilibrium of Lys25-ribonuclease T1 is adequately described by a simple two-state model. Upon unfolding the heat capacity increases by delta Cp degrees = 5.1 +/- 0.5 kJ/(mol.K). Similar values have been found for the unfolding of other small proteins. Surprisingly, this denaturational heat capacity change practically vanishes in the presence of moderate NaCl concentrations. The molecular origin of this effect is not clear; it is not observed to the same extent in the unfolding of bovine pancreatic ribonuclease A, which was employed in control experiments. NaCl stabilizes Lys25-ribonuclease T1. The transition temperature varies with NaCl activity in a manner that suggests two limiting binding equilibria to be operative. Below approximately 0.2 M NaCl activity unfolding is associated with dissociation of about one ion, whereas above that concentration about four ions are released in the unfolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

The refolding of ribonuclease T1 is dominated by two major slow kinetic phases that show properties of proline isomerization reactions. We report here that the molecular origin of one of these processes is the trans----cis isomerization of the Ser54-Pro55 peptide bond, which is cis in the native protein but predominantly trans in unfolded ribonuclease T1. This is shown by a comparison of the wild type and a designed mutant protein where Ser54 and Pro55 were replaced by Gly54 and Asn55, respectively. This mutation leaves the thermal stability of the protein almost unchanged; however, in the absence of Pro55 one of the two slow phases in folding is abolished and the kinetic mechanism of refolding is dramatically simplified.

It is our aim to elucidate molecular aspects of the mechanism of protein folding. We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds. Also, an efficient mutagenesis system is available to produce protein molecules with defined sequence variations. Here we present a preliminary characterization of the folding kinetics of ribonuclease T1. Its unfolding and refolding reactions are reversible, which is shown by the quantitative recovery of the catalytic activity after an unfolding/refolding cycle. Refolding is a complex process, where native protein is formed on three distinguishable pathways. There are 3.5% fast-folding molecules, which refold within the millisecond time range, and 96.5% slow-folding species, which regain the native state in the time range of minutes to hours. These slow-folding molecules give rise to two major, parallel refolding reactions. The mixture of fast- and slow-folding molecules is produced slowly after unfolding by chain equilibration reactions that show properties of proline isomerization. We conclude that part of the kinetic complexity of RNase T1 folding can be explained on the basis of the proline model for protein folding. This is supported by the finding that the slow refolding reactions of this protein are accelerated in the presence of the enzyme prolyl isomerase. However, several properties of ribonuclease T1 refolding, such as the dependence of the relative amplitudes on the probes, used to follow folding, are not readily explained by a simple proline model.

The slow refolding of ribonuclease T1 was investigated by different probes. Structural intermediates with secondary structure are formed early during refolding, as indicated by the rapid regain of a native-like circular dichroism spectrum in the amide region. This extensive structure formation is much faster than the slow steps of refolding, which are limited in rate by the reisomerization of incorrect proline isomers. The transient folding intermediates were also detected by unfolding assays, which make use of the reduced stability of folding intermediates relative to that of the native protein. The results of this and the preceding paper [Kiefhaber et al. (1990) Biochemistry (preceding paper in this issue)] were used to propose kinetic models for the unfolding and refolding of ribonuclease T1. The unfolding mechanism is based on the assumption that, after the structural unfolding step, the slow isomerizations of two X-Pro peptide bonds occur independently of each other in the denatured protein. At equilibrium a small amount of fast-folding species coexists with three slow-folding species: two with one incorrect proline isomer each and another, dominant species with both these prolines in the incorrect isomeric state. In the mechanism for refolding we assume that all slow-folding molecules can rapidly regain most of the secondary and part of the tertiary structure early in folding. Reisomerizations of incorrect proline peptide bonds constitute the slow, rate-limiting steps of refolding. A peculiar feature of the kinetic model for refolding is that the major unfolded species with two incorrect proline isomers can enter two alternative folding pathways, depending on which of the two reisomerizes first. The relative rates of reisomerization of the respective proline peptide bonds at the stage of the rapidly formed intermediate determine the choice of pathway. It is changed in the presence of prolyl isomerase, because this enzyme catalyzes these two isomerizations with different efficiency and consequently leads to a shift from the very slow to the intermediate refolding pathway.

Ribonuclease T1 (RNase-T1) from Aspergillus Oryzae cleaves ribonucleic acid specifically at guanosine to yield oligonucleotides with terminal guanosine-3'-phosphate. It forms a complex with vanadate (association constant K approximately 145 +/- 30 M-1; delta (51V) = -514 ppm) with spectral features similar to the less stable complexes obtained with di- and tripeptides (Gly-His, Pros-His-Ala, Gly-His-Lys, Val-Glu) containing amino acids that are constituents at the active site of the enzyme. Guanosine also forms a (sparingly soluble) complex with vanadate. Its role is mimicked by inosine, which yields two soluble complexes with vanadate, characterized by delta values of -511 (K = 94 M-1) and -523 ppm (K = 305 M-1 in TRIS buffer and 685 m-1 in buffer-free solution). Comparison with literature values leads to an assignment of the delta = -523 signal to a complex where monovanadate, possibly in a trigonal bipyramidal geometry suggested for the transition state of the phosphate analogue, is coordinated to the 2'- and 3'-oxygens of the ribose ring. A drastic increase of complex stability is observed in the ternary vanadate (12-16 mM)/inosine(10.5 mM)/RNase-T1(5.4 mM) system. An approximate lower limit for the association constant is 1.5.10(5) M-2. The spectral characteristics of the main component of the binary vanadate/inosine complex are essentially maintained (delta = -525 ppm, half-width = 960 Hz), suggesting vanadate binding to the enzyme through hydrogen bonds.

The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E. coli strain and ribonuclease T1 isolated from A. oryzae.

Studies using time-resolved fluorescence depolarization were performed on the internal motion of Trp 59 of ribonuclease T1 (EC 3.1.27.3) in the free enzyme, 2'-GMP-enzyme complex and 3'-GMP-enzyme complex. The Trp 59 motion was also studied in the free enzyme using molecular dynamics simulations. Energetic analysis of activation barriers to the Trp 59 motion was performed using both the transition state theory and Kramers' theory. The activation parameters showed a dependence on solvent viscosity indicating the transition state approach in aqueous solution to be inadequate. When taking solvent viscosity contributions into account agreement between the transition state and Kramers' theories was obtained. The results indicate the three enzyme forms to have different conformations with the free enzyme and 3'-GMP-enzyme complex being similar. Comparison of the experimental and theoretical results showed a good agreement on the Trp 59 motion in the free enzyme. Trp 59 appears to vibrate rapidly, with a relaxation time of the order of 1 ps, within free space in the protein matrix and to have a slower motion, with a relaxation time of the order of 100 ps, which is related to breathing of the surrounding protein matrix. Molecular dynamics results indicate high mobility in regions of the enzyme involved in the interaction with the guanine base of the inhibitor or substrate while much lower mobility occurred in residues involved in the catalytic mechanism of ribonuclease T1.