Effect of mutagenic replacement of the carboxyl terminal amino acid, val124, on the properties and regeneration of bovine pancreatic ribonuclease A

An important role of C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) in the formation of the three-dimensional structure was previously implied. In this study, we replaced the C-terminal amino acid, Val124, with amino acid residues with different properties by site-directed mutagenesis. The recombinant mutant enzymes were purified and subjected to a refolding study after being converted to a fully reduced and denatured state. There was a significant difference among the mutant enzymes in the rate of recovery of the activity when air oxidation was performed: the rate decreased in the order of V124E, V124L, V124G, V124K, V124A, and V124W. On the other hand, the recovery rates for all the mutant RNase A in the presence of GSH and GSSG were almost the same. The recovered activity of V124E after 24 h incubation reached approximately 90% of that of the wild type enzyme, followed by V124L 80%, V124A and V124W 65%, and V124K and V124G 50%. The duration of the initial lag phase became shorter in the order of V124W, V124A, V124K or V124G, V124E, or V124L. The results imply that the C-terminal amino acid significantly influences the formation of correct disulfide bonds during the refolding process and that the hydrophobic interaction of Val124 is important for efficient packing of the RNase A molecule.     

Multiple conformations of amino acid residues in ribonuclease A

The highly refined 1.26 A structure (R = 0.15) of phosphate-free bovine pancreatic ribonuclease A was modeled with 13 residues having discrete multiple conformations of side chains. These residues are widely distributed over the protein surface, but only one of them, Lys 61, is involved in crystal packing interactions. The discrete conformers have no unusual torsion angles, and their interactions with the solvent and with other atoms of the protein are similar to those residues modeled with a single conformation. For three of the residues--Val 43, Asp 83, and Arg 85--two correlated conformations are found. The observed multiple conformations on the protein surfaces will be of significance in analyzing structure-function relationships and in performing protein engineering.     

Catalysis of the oxidative folding of bovine pancreatic ribonuclease A by protein disulfide isomerase

The major oxidative folding pathways of bovine pancreatic ribonuclease A at pH 8.0 and 25 degrees C involve a pre-equilibrium steady state among ensembles of intermediates with zero, one, two, three and four disulfide bonds. The rate-determining steps are the reshuffling of the unstructured three-disulfide ensemble to two native-like three-disulfide species, des-[65-72] and des-[40-95], that convert to the native structure during oxidative formation of the fourth disulfide bond. Under the same regeneration conditions, with oxidized and reduced DTT, used previously for kinetic oxidative-folding studies of this protein, the addition of 4 microM protein disulfide isomerase (PDI) was found to lead to catalysis of each disulfide-formation step, including the rate-limiting rearrangement steps in which the native-like intermediates des-[65-72] and des-[40-95] are formed. The changes in the distribution of intermediates were also determined in the presence and absence of PDI at three different temperatures (with the DTT redox system) as well as at 25 degrees C (with the glutathione redox system). The results indicate that the acceleration of the formation of native protein by PDI, which we observed earlier, is due to PDI catalysis of each of the intermediate steps without changing the overall pathways or folding mechanism.     

Modification of amino acids and bovine pancreatic ribonuclease A by kethoxal.

Kethoxal (3-ethoxy-2-ketobutanal) reacts with the guanidino group of Nalpha-acetylarginine to produce four derivatives, reactive to periodate, stable at pH 7, with 15% reverting to arginine on acid hydrolysis. Other amino acids with blocked alpha-amino groups do not react, except the epsilon-amino of lysine (slowly). The pK of the mixed Kethoxal-Nalpha-acetylarginine derivatives is 5.8-6.1. Kethoxal reacts at neutral pH with arginyl residues of bovine pancreatic ribonuclease A. In the presence of an active-site ligand, arginine-39 and arginine-85 react at about equal rates. The loss of enzymic activity at pH 7 is proportional to the combined loss of these residues. The enzymic activity toward RNA is 20-25% of that of native RNAase at pH 7, and 90-100% at pH 5. In the absence of an active site ligand, arginine-10 is also modified with the loss of almost all enzymic activity, although arginine-10 is not an active-site residue. Arginine-33 is unreactive. Kethoxal-modified RNAase undergoes cross-linking in solution at pH 7 or in the freeze-dried state, Incubation at pH 9 in the presence of homoarginine results in partial regeneration of arginyl residues and activity at pH 7. Kethoxal modification of arginines-39 and -85 appears to raise the pK of lysine-41 by about 1 unit, as indicated ty the pH dependence of arylation by 2-carboxy-4,6-dinitrochlorobenzene. The claims of Patthy and Smith (J. Biol, Chem. (1975) 250, 565-569), and of Takahashi (J. Biol. Chem. (1968) 243, 6171-6179) that arginine-39 is a more important functional residue than is arginine-85 are questioned.     

Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A.

The Tyr92-Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfide-intact wild-type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92-Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C[40, 95]A variant, which is an analog of the major rate-determining three-disulfide intermediate in the oxidative folding of RNase A, missing the 40-95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.     

The amino acid sequence of hamster pancreatic ribonuclease

The amino acid sequence of golden hamster pancreatic ribonuclease was determined by analysis of tryptic, chymotryptic, thermolytic, and CNBr peptides and by automatic sequence analysis of the intact protein. Like all RNases with an Asn-Met-Thr sequence at positions 34-36, hamster RNase is glycosylated at position 34 with a complex-type carbohydrated chain. Val-17, Ala-18, His-55, His-76 and Ala-90 have never been observed in other pancreatic RNases. Ala-90 replaces Ser-90, which had been invariant in all mammalian RNases studied so far. The amino acid sequence of hamster RNase differs at 15 positions from that of another Cricetidae rodent, the muskrat. The similarity between both ribonucleases was used to confirm a few less certain parts of the muskrat RNase sequence. The replacement rate of the RNases of the Cricetidae appeared to be higher than the average rate in the mammals, but much lower than the rate in another myomorph family, the Muridae (mouse and rat). Possibly, in many respects, the Cricetidae underwent less evolutionary change in recent times than the evolutionarily highly successful Muridae.     

Structural studies of a folding intermediate of bovine pancreatic ribonuclease A by continuous recycled flow

A new technique, continuous recycled flow (CRF) spectroscopy, has been developed for observing intermediates of any thermally induced, reversible reaction with a half-life of 10 s or longer. The structure can be probed by any spectroscopic method which does not perturb the system. Prolonged signal acquisitions of 8 h for ribonuclease A are possible. CRF was used to investigate the structure of the slow-folding intermediates of chemically intact ribonuclease A (RNase A) during thermal unfolding/folding under acidic conditions. The following conclusions were reached on the basis of the proton nuclear magnetic resonance and far-ultraviolet circular dichroism spectra of a folding intermediate(s): (A) The conformation of the detected folding intermediate(s) is similar to that of the heat-denatured protein. There is only limited formation of new structures. (B) The N-terminal alpha-helix is partially stable under these conditions and is in rapid (less than 10 ms) equilibrium with the denatured conformation. (C) There are long-range interactions between the hydrophobic residues of the N-terminal alpha-helix and the rest of the protein. These interactions persist well above the melting point. (D) An aliphatic methyl group reports on the formation of a new structure(s) that lie(s) outside of the N-terminal region. (E) The structures detected in chemically modified, nonfolding forms of the RNase A are also present in the folding intermediate(s). There are, however, additional interactions that are unique to chemically intact RNase A.     

Interactions that favor the native over the non-native disulfide bond among residues 58-72 in the oxidative folding of bovine pancreatic ribonuclease A

In the initial stages of the oxidative folding of both bovine pancreatic ribonuclease A (RNase A) and a 58-72 fragment thereof from the fully reduced, denatured state, the 65-72 correctly paired disulfide bond forms in preponderance over the incorrectly paired 58-65 disulfide bond. Since both disulfide-bonded loops contain the same number of amino acid residues, the question arises as to whether the native pairing results from interactions within the 58-72 segment that lead to a nativelike structure even in its fully reduced form. To answer this question, the chain buildup procedure, based on ECEPP, including a solvation treatment, was used to generate the low-energy structures for the 58-72 RNase segment, beginning with residue 72 and building back to residue 58; in this fragment, all three Cys residues (at positions 58, 65, and 72) initially exist in the reduced (CysH) state. After the open-chain energy minima of the 65-72 peptide were generated, these conformations were allowed to form the 65-72 disulfide bond, and the energies of the resulting oxidized conformations were reminimized and rehydrated. The global minimum of the loop-closed 65-72 structure and many of the low-lying loop-closed minima could be superimposed on the energy-minimized X-ray structure for residues 65-72. The low-energy structures for the full open chain 58-72 peptide were then computed and were allowed to form disulfide bonds either between residues 65 and 72 (native) or between residues 58 and 65 (non-native), and their energies were reminimized and rehydrated in the loop-closed state. Although the overall fold of the 65-72 loop-closed global minimum was the same as for the energy-minimized X-ray structure of these residues, the overall rms deviation was 3.9 A because of local deviations among residues 58-64. In contrast, the 65-72 segment of the global minimum of the 58-72 fragment could be superimposed on the corresponding residues of the energy-minimized X-ray structure. The lowest-energy structure for the 58-65 non-native paired 58-72 sequence was 6 kcal/mol higher in energy than that for the 58-72 peptide with the 65-72 disulfide bond formed. These results suggest that the native pairing of the 65-72 peptide arises from energetic determinants (adoption of left-handed single-residue conformations by Gly 68, and side chain interactions involving Gln 69) contained within this peptide sequence.     

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.     

Effect of protein disulfide isomerase on the rate-determining steps of the folding of bovine pancreatic ribonuclease A

To address the effect of an agglutogen on virus infection, we studied the avidin-associated inhibition of infection by biotinylated M13 phages (BIO-phages). Microscopic observation of mixtures of BIO-phages and avidin–fluorescein conjugates revealed many aggregates. Even at low phage concentrations, avidin induced inhibition of infection significantly. Anti-M13 phage antibody also made aggregates and inhibited the infection but in a different manner from avidin. The inhibition by avidin was at ≥2 μg/ml, time dependent and marked until 10 min after the mixing of the BIO-phages and Escherichia coli. On the other hand, antibody inhibited the infection at ≥0.1 μg/ml dose dependently, and the inhibition was time dependent and marked until 45 min after the mixing at moderate and low phage concentrations. These results indicate that avidin against BIO-phages and antibodies are agglutogens, and the inhibition of the BIO-phages by avidin is closely related to the tetramerization of avidin. Agglutogens may be novel alternative antiviral drugs.     

Thermal cycling aids folding of a recombinant human beta-casein with four extra N-terminal amino acid residues

Due to the limited secondary structure, it is believed that the caseins of milk, particularly the beta-caseins (beta-CN), may be in a mostly random-coil conformation or in various structures that result from random association of hydrophobic residues. However, the self-association of the human proteins with increasing temperature (T) and in the presence of Ca2+ is reproducible, implying that they normally fold into fixed tertiary structures. A nonphosphorylated recombinant human beta-CN with four extra amino acids at the N-terminus (GSHM-) was prepared and studied by laser light scattering, analytical ultracentrifugation, fluorescence spectroscopy, turbidity, and circular dichroism. In 3.3 M urea or at 4 degrees C, the protein was monomeric, as expected. Increasing T both without and with the addition of Ca2+ ions caused self-association as it does for the nonphosphorylated native beta-CN but with a somewhat different interaction pattern. However, returning the protein to its monomeric state by reequilibration at 4 degrees C followed again by increasing T caused a shift in the pattern. Such thermal cycling eventually caused the protein to equilibrate to a particular conformation where no more change could be observed. The resulting interaction pattern was similar to that of the native protein but differed particularly in that there was more extensive self-association for the recombinant mutant. The equilibration to a stable conformation was more rapid in the presence of Ca2+ ions. This suggests that the native protein normally folds into a particular conformation which may be aided by Ca2+ in the mammary gland. Further study of a recombinant form with the native amino acid sequence is needed.