Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribonuclease T1 has two disulfide bonds linking cysteine residues 2-10 and 6-103. We have prepared a derivative of ribonuclease T1 in which one disulfide bond is broken and the cysteine residues carboxymethylated, (2-10)-RCM-T1, and three derivatives in which both disulfides are broken and the cysteine residues reduced, R-T1, carboxamidomethylated, RCAM-T1, or carboxymethylated, RCM-T1. The RNA hydrolyzing activity of these proteins has been measured, and urea and thermal denaturation studies have been used to determine conformational stability. The activity, melting temperature, and conformational stability of the proteins are: ribonuclease T1 (100%, 59.3 degrees C, 10.2 kcal/mol), (2-10)-RCM-T1 (86%, 53.3 degrees C, 6.8 kcal/mol), R-T1 (53%, 27.2 degrees C, 3.0 kcal/mol), RCAM-T1 (43%, 21.2 degrees C, 1.5 kcal/mol), and RCM-T1 (35%, 16.6 degrees C, 0.9 kcal/mol). Thus, the conformational stability is decreased by 3.4 kcal/mol when one disulfide bond is broken and by 7.2-9.3 kcal/mol when both disulfide bonds are broken. It is quite remarkable that RNase T1 can fold and function with both disulfide bonds broken and the cysteine residues carboxymethylated. The large decrease in the stability is due mainly to an increase in the conformational entropy of the unfolded protein which results when the constraints of the disulfide bonds on the flexibility are removed. We propose a new equation for predicting the effect of a cross-link on the conformational entropy of a protein: delta Sconf = -2.1 - (3/2)R 1n n, where n is the number of residues between the side chains which are cross-linked. This equation gives much better agreement with experimental results than other forms of this equation which have been used previously.
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PMID:Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds. 245 27

1. RNase Ms, a base non-specific RNase from Aspergillus saitoi was reduced and carboxymethylated (RCM-RNase Ms). RCM-RNase Ms was hydrolyzed with trypsin, and the trypsin digests were then treated with chymotrypsin. Trypsin digests were also treated with Staphylococcus protease and with chymotrypsin, separately. 2. By the analyses of the amino acid sequences of the peptides formed, the alignment of these peptides in RCM-RNase Ms was determined. 3. From the digest of heat-denatured RNase Ms with Bacillus subtilis protease, two peptides containing disulfide bridges were isolated. From the analysis of these two peptides, the locations of the bridges were determined. 4. The amino acid sequence of RNase Ms was compared with those of RNase T1 (Asp. oryzae, guanine specific), RNase U1 (Ustilago sphaerogena, guanine specific) and RNase U2 (Ustilago sphaerogena, purine specific). There are very similar sequences between these for RNases irrespective of their differences in base specificity. These were, in RNase Ms, tripeptide sequence containing His39 (Tyr-Pro-His), the tetrapeptide containing Glu57 (Glu-Tyr-Pro-Ile), the hexapeptide containing Arg76 (Asp-Arg-Val-Ile-Phe-Asp) and the hexapeptide containing His 91 (Ile-Thr-His-Thr-Gly-Ala). The other sequences common for all four RNases are Tyr67, Phe100, and Cys103 in RNase Ms. Since among these peptides His39, Glu57, His91, and Arg76 in RNase Ms corresponded to His40, Glu58, His92, and Arg77 in RNase T1 which are known to be involved in the active site of RNase T1, the possible role of these amino acids in the active site of RNase Ms is discussed. 5. The sequence similarity of RNase Ms to that of RNase T1 was about 60% and to those of RNase U1 and RNase U2 was about 30%. 6. The details of the experimental evidence used to elucidate the amino acid sequence of RNase Ms are described in the supplemental miniprint.
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PMID:Primary structure of a minor ribonuclease from Aspergillus saitoi. 709 2

SlyD is a putative folding helper protein from the Escherichia coli cytosol, which consists of an N-terminal prolyl isomerase domain of the FKBP type and a presumably unstructured C-terminal tail. We produced truncated versions without this tail (SlyD) for SlyD from E. coli, as well as for the SlyD orthologues from Yersinia pestis, Treponema pallidum, Pasteurella multocida, and Vibrio cholerae. They are monomeric in solution and unfold reversibly. All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies, and the specificity constants (kcat/KM) are equal to approximately 10(6) M(-1) s(-1). These large values originate from the high affinities of the SlyD orthologues for unfolded RCM-T1, which are reflected in low KM values of approximately 1 microM. SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl isomerase activity. The unfolded protein chains do not need to contain proline residues to be recognized and bound by SlyD. The conservation of prolyl isomerase activity and chaperone properties within the SlyD family suggests that these proteins might act as true folding helpers in the bacterial cytosol. The SlyD proteins are also well suited for biotechnological applications. As fusion partners they facilitate the refolding and increase the solubility of aggregation-prone proteins such as the gp41 ectodomain fragment of HIV-1.
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PMID:SlyD proteins from different species exhibit high prolyl isomerase and chaperone activities. 1638 77