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1.
Org Biomol Chem ; 14(37): 8804-8814, 2016 Sep 21.
Article in English | MEDLINE | ID: mdl-27714155

ABSTRACT

In this paper, we have used total chemical synthesis of RNase A analogues in order to probe the molecular basis of enzyme catalysis. Our goal was to obligately fill the adenine-binding pocket on the enzyme molecule, and to thus pre-orient the imidazole side chain of His119 in its catalytically productive orientation. Two designed analogues of the RNase A protein molecule that contained an adenine moiety covalently bound to distinct amino acid side chains adjacent to the adenine binding pocket were prepared. A crystal structure of one analogue was determined at 2.3 Å resolution. Kinetic data for RNA transphosporylation and 2',3' cyclic mononucleotide hydrolysis were acquired for the adenine-containing RNase A analogue proteins. As anticipated, the presence of a covalently attached adenine on the enzyme molecule decreased the rate of transphosphorylation and increased the rate of hydrolysis, although the magnitude of the effects was small. This work illustrates the use of total protein synthesis to investigate the chemistry of enzyme catalysis in ways not possible through traditional biochemistry or molecular biology.


Subject(s)
Ribonuclease, Pancreatic/chemical synthesis , Adenine/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cattle , Crystallography, X-Ray , Hydrolysis , Molecular Docking Simulation , Phosphorylation , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/metabolism
2.
Biopolymers ; 90(3): 278-86, 2008.
Article in English | MEDLINE | ID: mdl-17610259

ABSTRACT

The total chemical synthesis of RNase A using modern chemical ligation methods is described, illustrating the significant advances that have been made in chemical protein synthesis since Gutte and Merrifield's pioneering preparation of RNase A in 1969. The identity of the synthetic product was confirmed through rigorous characterization, including the determination of the X-ray crystal structure to 1.1 Angstrom resolution.


Subject(s)
Ribonuclease, Pancreatic/analysis , Ribonuclease, Pancreatic/chemical synthesis , Amino Acid Sequence , Animals , Catalysis , Cattle , Cysteine/chemistry , Disulfides/chemistry , Hydrogen Bonding , Kinetics , Models, Chemical , Molecular Sequence Data , Protein Conformation , Protein Folding , Protein Structure, Secondary , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/isolation & purification , Water/chemistry , X-Ray Diffraction
3.
Biochemistry ; 43(8): 2167-77, 2004 Mar 02.
Article in English | MEDLINE | ID: mdl-14979713

ABSTRACT

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.


Subject(s)
Growth Inhibitors/chemical synthesis , Growth Inhibitors/toxicity , Nuclear Localization Signals/chemical synthesis , Nuclear Localization Signals/toxicity , Ribonuclease, Pancreatic/chemical synthesis , Ribonuclease, Pancreatic/toxicity , Active Transport, Cell Nucleus/genetics , Catalysis , Cell Nucleus/enzymology , Cell Nucleus/genetics , Drug Design , Endocytosis/genetics , Enzyme Inhibitors/chemistry , Enzyme Stability/genetics , Genetic Variation , Growth Inhibitors/antagonists & inhibitors , Growth Inhibitors/genetics , HeLa Cells , Humans , Inhibitory Concentration 50 , Intracellular Fluid/enzymology , Isoenzymes/biosynthesis , Isoenzymes/chemical synthesis , Isoenzymes/genetics , Isoenzymes/toxicity , K562 Cells , Mutagenesis, Site-Directed , Nuclear Localization Signals/biosynthesis , Nuclear Localization Signals/genetics , Ribonuclease, Pancreatic/antagonists & inhibitors , Ribonuclease, Pancreatic/genetics
4.
J Am Chem Soc ; 125(18): 5268-9, 2003 May 07.
Article in English | MEDLINE | ID: mdl-12720426

ABSTRACT

Chemical synthesis harbors the potential to provide ready access to natural proteins as well as to create nonnatural ones. The Staudinger ligation of a peptide containing a C-terminal phosphinothioester with a peptide containing an N-terminal azide gives an amide with no residual atoms. This method for amide bond formation is orthogonal and complementary to other ligation methods. Herein, we describe the first use of the Staudinger ligation to couple peptides on a solid support. The fragment thus produced is used to assemble functional ribonuclease A via native chemical ligation. The synthesis of a protein by this route expands the versatility of chemical approaches to protein production.


Subject(s)
Peptide Fragments/chemical synthesis , Ribonuclease, Pancreatic/chemical synthesis , Amino Acids/chemistry , Kinetics , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/metabolism
5.
J Am Chem Soc ; 124(29): 8522-3, 2002 Jul 24.
Article in English | MEDLINE | ID: mdl-12121081

ABSTRACT

beta-Amino acids are incorporated into an enzyme by using the method of expressed protein ligation. In the resulting semisynthetic enzyme, an R-nipecotic acid-S-nipecotic acid module replaces Asn113 and Pro114 of ribonuclease A. The semisynthetic enzyme not only retains full catalytic activity but also gains conformational stability. Thus, structural elements can be replaced with foldameric equivalents to endow proteins with more desirable properties.


Subject(s)
Ribonuclease, Pancreatic/chemical synthesis , Catalysis , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Ribonuclease, Pancreatic/chemistry
7.
Protein Sci ; 6(10): 2233-41, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9336846

ABSTRACT

We have studied the conformation as well as V8 protease-mediated synthesis of peptide fragments, namely amino acid residues 295-316 (TC-peptide) of thermolysin and residues 1-20 (S-peptide) of ribonuclease A, to examine whether "conformational trapping" of the product can facilitate reverse proteolysis. The circular dichroism study showed cosolvent-mediated cooperative helix formation in TC-peptide with attainment of about 30-35% helicity in the presence of 40% 1-propanol and 2-propanol solutions at pH 6 and 4 degrees C. The thermal melting profiles of TC-peptide in the above cosolvents were very similar. V8 protease catalyzed the synthesis of TC-peptide from a 1:1 mixture of the non-interacting complementary fragments (TC295-302 and TC303-316) in the presence of the above cosolvents at pH 6 and 4 degrees C. In contrast, V8 protease did not catalyze the ligation of S1-9 and S10-20, although S-peptide could assume helical conformation in the presence of the cosolvent used for the semisynthetic reaction. V8 protease was able to synthesize an analog of S-peptide (SA-peptide) in which residues 10-14 were substituted (RQHMD-->VAAAK). While S-peptide exhibited helical conformation in the presence of aqueous propanol solutions, SA-peptide displayed predominantly beta-sheet conformation. SA-peptide showed enhanced resistance to proteolysis as compared with S-peptide. Thus, failure of semisynthesis of S-peptide may be a consequence of high flexibility around the 9-10 peptide bond due to its proximity to the helix stop signal. The results suggest that protease-mediated ligations may be achieved by design and manipulation of the conformational aspects of the product.


Subject(s)
Peptide Fragments/chemical synthesis , Protein Conformation , Ribonuclease, Pancreatic/chemical synthesis , Serine Endopeptidases/metabolism , Thermolysin/chemical synthesis , 1-Propanol , Amino Acid Sequence , Circular Dichroism , Crystallization , Hydrogen-Ion Concentration , Molecular Sequence Data , Protein Structure, Secondary , Solutions , Temperature
8.
Science ; 266(5183): 243-7, 1994 Oct 14.
Article in English | MEDLINE | ID: mdl-7939659

ABSTRACT

An engineered variant of subtilisin BPN', termed subtiligase, which efficiently ligates esterified peptides in aqueous solution, was used for the complete synthesis of ribonuclease (RNase) A that contains unnatural catalytic residues. Fully active RNase A (124 residues long) was produced in milligram quantities by stepwise ligation of six esterified peptide fragments (each 12 to 30 residues long) at yields averaging 70 percent per ligation. Variants of RNase A were produced in which the catalytic histidines at positions 12 and 119 were substituted with the unnatural amino acid 4-fluorohistidine, which has a pKa of 3.5 compared to 6.8 for histidine. Large changes in the profile of the pH as it affects rate occurred for the single and double mutants with surprisingly little change in the kcat for either the RNA cleavage or hydrolysis steps. The data indicate that these imidazoles function as general acids and bases, but that the proton transfer steps are not rate-limiting when the imidazoles are present in their correct protonation states. These studies indicate the potential of subtiligase for the blockwise synthesis of large proteins.


Subject(s)
Protein Engineering/methods , Ribonuclease, Pancreatic/chemical synthesis , Subtilisins/metabolism , Amino Acid Sequence , Binding Sites , Esterification , Histidine/analogs & derivatives , Histidine/analysis , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutation , Nucleotides, Cyclic/metabolism , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/isolation & purification , Subtilisins/chemistry , Subtilisins/genetics , Uridine Monophosphate/metabolism
9.
Protein Sci ; 3(2): 248-56, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8003961

ABSTRACT

Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamido-contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1, Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol-1. Modifications that introduced a positive charge in position 7 (S-aminoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-1 loss in binding energy. S-protein bound to RI with Ki = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol-1 difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.


Subject(s)
Proteins/metabolism , Ribonuclease, Pancreatic/chemical synthesis , Ribonuclease, Pancreatic/metabolism , Ribonucleases/antagonists & inhibitors , Animals , Catalysis , Chromatography, High Pressure Liquid , Circular Dichroism , Hydrogen Bonding , Intracellular Signaling Peptides and Proteins , Kinetics , Liver/enzymology , Osmolar Concentration , Peptide Fragments/metabolism , Protein Binding , RNA, Fungal/metabolism , Recombinant Proteins/metabolism , Ribonuclease, Pancreatic/chemistry , Structure-Activity Relationship , Substrate Specificity , Subtilisins/metabolism , Swine , Thermodynamics
10.
Nucleic Acids Res ; 21(25): 5950-6, 1993 Dec 25.
Article in English | MEDLINE | ID: mdl-7507235

ABSTRACT

RNA cleaving molecules were synthesized by conjugating imidazole residues imitating the essential imidazoles in the active center of pancreatic ribonuclease to an intercalating compound, derivative of phenazine capable of binding to the double stranded regions of polynucleotides. Action of the molecules on tRNA was investigated. It was found, that some of the compounds bearing two imidazole residues cleave tRNA under physiological conditions. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0 indicating participation of protonated and non-protonated imidazole residues in the process. Under the conditions stabilizing the tRNA structure, a tRNAAsp transcript was cleaved preferentially at the junctions of the stem and loop regions of the cloverleaf tRNA fold, at the five positions C56, C43, C20.1, U13, and U8, with a marked preference for C56. This cleavage pattern is consistent with a hydrolysis mechanism involving non-covalent binding of the compounds to the double-stranded regions of tRNA followed by an attack of the imidazole residues at the juxtaposed flexible single-stranded regions of the molecule. The compounds provide new probes for the investigation of RNA structure in solution and potential reactive groups for antisense oligonucleotide derivatives.


Subject(s)
RNA, Transfer, Asp/metabolism , Ribonuclease, Pancreatic/metabolism , Base Sequence , Binding Sites , Hydrogen-Ion Concentration , Molecular Sequence Data , Nucleic Acid Conformation , RNA/metabolism , Ribonuclease, Pancreatic/antagonists & inhibitors , Ribonuclease, Pancreatic/chemical synthesis , Substrate Specificity , Temperature , Transcription, Genetic
11.
Vopr Med Khim ; 36(5): 4-6, 1990.
Article in Russian | MEDLINE | ID: mdl-2251790

ABSTRACT

Conjugates containing blood serum albumin and pancreatic ribonuclease, produced by means of polycondensation reaction, exhibited higher half-life in rabbit circulation as compared with non-modified enzyme. Presence of the protein-carrier contributed to elevation of the ribonuclease therapeutic efficiency and enabled to decrease the quantity of injections.


Subject(s)
Ribonuclease, Pancreatic/chemistry , Serum Albumin/chemistry , Animals , Delayed-Action Preparations , Drug Carriers , Half-Life , Rabbits , Ribonuclease, Pancreatic/administration & dosage , Ribonuclease, Pancreatic/chemical synthesis , Ribonuclease, Pancreatic/pharmacokinetics , Serum Albumin/chemical synthesis
12.
J Biol Chem ; 262(33): 15930-8, 1987 Nov 25.
Article in English | MEDLINE | ID: mdl-3680234

ABSTRACT

A fully active, semisynthetic analog of bovine ribonuclease A, comprised of residues 1-118 of the molecule in a noncovalent complex with the synthetic peptide analog of residues 111-124, has been crystallized in space group P3(2)21 from a solution of 1.3 M ammonium sulfate and 3.0 M cesium chloride at pH 5.2. The crystallographic structure was determined by rotation and translation searches utilizing the coordinates for ribonuclease A reported by Wlodawer and Sjolin (Wlodawer, A., and Sjolin, L. (1983) Biochemistry 22, 2720-2728) and has been refined at 1.8-A resolution to an agreement factor of 0.204. Most of the structure of the semisynthetic enzyme closely resembles that found in ribonuclease A with the synthetic peptide replacing the C-terminal elements of the naturally occurring enzyme. No redundant structure is seen; residues 114-118 of the larger chain and residues 111-113 of the peptide do not appear in our map. The positions of those residues at or near the active site are very similar to, if not identical with, those previously reported by others, except for histidine 119, which occupies predominantly the B position seen as a minor site by Borkakoti et al. (Borkakoti, N., Moss, D. S., and Palmer, R. A. (1982) Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 38,2210-2217) and not at all by Wlodawer and Sjolin (1983).


Subject(s)
Ribonuclease, Pancreatic , Amino Acid Sequence , Animals , Cattle , Crystallization , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Protein Conformation , Ribonuclease, Pancreatic/chemical synthesis , Solutions , X-Ray Diffraction
17.
Int J Pept Protein Res ; 23(1): 61-71, 1984 Jan.
Article in English | MEDLINE | ID: mdl-6698716

ABSTRACT

The S-peptide of the enzyme bovine pancreatic ribonuclease has been used as a model for covalent semisynthesis. Methods for side-chain protection, enzymatic cleavage of the peptide chain at the level of the single arginine-10 and for selective deprotection of the alpha-carboxyl function of this residue, have been examined. The partially protected [1-10] sequence has been coupled to a solid-phase generated [11-15] sequence attached to the polymer. After deblocking from the solid-support, the [1-15] semisynthetic peptide was complexed with native S-protein to give a complex with high biological activity.


Subject(s)
Peptide Fragments/chemical synthesis , Ribonuclease, Pancreatic/chemical synthesis , Amino Acid Sequence , Animals , Arginine , Cattle , Indicators and Reagents , Ribonuclease, Pancreatic/metabolism , Trypsin
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