Structure of phosphate-free ribonuclease A refined at 1.26 .ANG.

Wlodawer, Alexander; Svensson, L.A.; Sjölin, L.; Gilliland, Gary L.

doi:10.1021/bi00408a010

Cited by 382 publications

(414 citation statements)

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“…The crystal structure used here is the best-resolved crystal structure of this study with a resolution of 1-26 A and an R-factor of 0-15 (Wlodawer et al 1988). Another high-resolution crystal structure was reported earlier by Borkakoti et al (1982;resolution: 1-45 A); differences between these two crystal structures reveal available at https:/www.cambridge.org/core/terms.…”

Section: Ribonucleasementioning

confidence: 62%

Comparison of protein structures determined by NMR in solution and by X-ray diffraction in single crystals

Billeter¹

1992

Quart. Rev. Biophys.

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Following the first determinations of protein structures in the late 1950s and the early 1960s (see for example Kendrewet al.1960; Perutz, 1964), the three-dimensional structures of several hundred proteins have been elucidated by X-ray diffraction on single crystals. By the end of 1991, approximately 150 entries of proteins with substantially different sequences and a well resolved structure (Hobohmet al.1992) were deposited in the Protein Data Bank (Bernsteinet al.1977; Abolaet al.1987). In addition, many structures of homologous proteins or of mutants have been described, bringing the total number of entries to about 600. While it was soon accepted that almost all of these structures do indeed give a correct picture of the fold of the active protein in spite of the non-physiological environment of single crystals, it is not clear to what extent structural details are reliably described by these structures. In particular the surface of a protein may be modified due to the dense packing of protein molecules in the crystal lattice. A detailed knowledge of the protein surface is, however, essential for the understanding of the function of the protein.

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Section: Ribonucleasementioning

confidence: 62%

Comparison of protein structures determined by NMR in solution and by X-ray diffraction in single crystals

Billeter¹

1992

Quart. Rev. Biophys.

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“…In the wild-type monoclinic structure, N ε2 of Gln11 forms a 3.0 Å hydrogen bond with a conserved water molecule in the active site, but O ε1 forms no hydrogen bonds to surrounding water molecules (48). In the structure of D121N RNase A, the sidechain of Gln11 adopts a slightly different conformation.…”

Section: Gln11mentioning

confidence: 99%

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

1998

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The sidechains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His⋯Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His⋯Asp dyad is composed of His119 and Asp121. Previously, sitedirected mutagenesis was used to show that His119 has a fundamental role-to act as an acid during catalysis of RNA cleavage , J. Am. Chem. Soc. 116,[5467][5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 Å with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, N δ rather than O δ of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3′→5′)adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2′,3′-cyclic phosphate. Replacing Asp121 decreases the values of k cat /K m and k cat for cleavage by 101-fold (D121N) and 10 2 -fold (D121A). Replacing Asp121 also decreases the values of k cat /K m and k cat for hydrolysis by 10 0.5 -fold (D121N) and 10-fold (D121A), but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His⋯Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.Keywords catalytic dyad; catalytic triad; low-barrier hydrogen bond; pH-rate profile; ribonuclease A; serine protease; site-directed mutagenesis; X-ray crystallographyThe amino acid motifs available to enzymic catalysts are diverse. Still, many enzymes fall into classes wherein great similarities exist. One well-studied class is that of the serine proteases (1). This class of enzymes has an active-site motif known as the catalytic triad, which is composed of the residues Ser⋯His⋯Asp linked by hydrogen bonds (2,3). † This work was supported by Grant GM44783 (NIH). X-ray data collection and computational facilities were supported by Grant BIR-9317398 (NSF). L.W.S. was supported by postdoctoral fellowship CA69750 (NIH). D.J.Q. was supported by Cellular and Molecular Biology Training Grant GM07215 (NIH).*Author to whom correspondence should be addressed.. ‡ Present address: School of Pharmacy, University of Wisconsin -Madison, Madison, WI 53706. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2...

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“…Structural properties of RNase dissolved in aqueous TFE that likely contribute to its specific nicking by thermolysin are discussed. Conformational and functional properties of the two nicked RNase molecules are interpreted, taking advantage of the known three-dimensional structure and dynamics of native RNase in the crystal state (Wyckoff et al, 1970;Wlodawer et al, 1982Wlodawer et al, , 1988 or in solution (Rico et al, 1989;Santoro et al, 1993)? concentration.…”

mentioning

confidence: 99%

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

et al. 1997

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We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42"C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Thl) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Thl maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-ATm 16 "C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Thl is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Thl retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 3 1-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a &strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Thl and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.Keywords: CD; limited proteolysis; NMR; ribonuclease A; thermolysin; trifluoroethanol Short-chain alcohols, such as methanol, ethanol, propanol, chloroethanol, and, in particular, trifluoroethanol, have been used frequently to induce a-helical conformations in otherwise randomly coiled or partially structured polypeptides (Toniolo et al., 1975;

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Structure of phosphate-free ribonuclease A refined at 1.26 .ANG.

Cited by 382 publications

References 39 publications

Comparison of protein structures determined by NMR in solution and by X-ray diffraction in single crystals

Comparison of protein structures determined by NMR in solution and by X-ray diffraction in single crystals

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

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