Derivatives Of Ribonuclease Research Articles

Rigidity analysis of protein biological assemblies and periodic crystal structures

BackgroundWe initiate in silico rigidity-theoretical studies of biological assemblies and small crystals for protein structures. The goal is to determine if, and how, the interactions among neighboring cells and subchains affect the flexibility of a molecule in its crystallized state. We use experimental X-ray crystallography data from the Protein Data Bank (PDB). The analysis relies on an effcient graph-based algorithm. Computational experiments were performed using new protein rigidity analysis tools available in the new release of our KINARI-Web server http://kinari.cs.umass.edu.ResultsWe provide two types of results: on biological assemblies and on crystals. We found that when only isolated subchains are considered, structural and functional information may be missed. Indeed, the rigidity of biological assemblies is sometimes dependent on the count and placement of hydrogen bonds and other interactions among the individual subchains of the biological unit. Similarly, the rigidity of small crystals may be affected by the interactions between atoms belonging to different unit cells.We have analyzed a dataset of approximately 300 proteins, from which we generated 982 crystals (some of which are biological assemblies). We identified two types of behaviors. (a) Some crystals and/or biological assemblies will aggregate into rigid bodies that span multiple unit cells/asymmetric units. Some of them create substantially larger rigid cluster in the crystal/biological assembly form, while in other cases, the aggregation has a smaller effect just at the interface between the units. (b) In other cases, the rigidity properties of the asymmetric units are retained, because the rigid bodies did not combine.We also identified two interesting cases where rigidity analysis may be correlated with the functional behavior of the protein. This type of information, identified here for the first time, depends critically on the ability to create crystals and biological assemblies, and would not have been observed only from the asymmetric unit.For the Ribonuclease A protein (PDB file 5RSA), which is functionally active in the crystallized form, we found that the individual protein and its crystal form retain the flexibility parameters between the two states. In contrast, a derivative of Ribonuclease A (PDB file 9RSA), has no functional activity, and the protein in both the asymmetric and crystalline forms, is very rigid.For the vaccinia virus D13 scaffolding protein (PDB file 3SAQ), which has two biological assemblies, we observed a striking asymmetry in the rigidity cluster decomposition of one of them, which seems implausible, given its symmetry. Upon careful investigation, we tracked the cause to a placement decision by the Reduce software concerning the hydrogen atoms, thus affecting the distribution of certain hydrogen bonds. The surprising result is that the presence or lack of a very few, but critical, hydrogen bonds, can drastically affect the rigid cluster decomposition of the biological assembly.ConclusionThe rigidity analysis of a single asymmetric unit may not accurately reflect the protein's behavior in the tightly packed crystal environment. Using our KINARI software, we demonstrated that additional functional and rigidity information can be gained by analyzing a protein's biological assembly and/or crystal structure. However, performing a larger scale study would be computationally expensive (due to the size of the molecules involved). Overcoming this limitation will require novel mathematical and computational extensions to our software.

Experimental evidence for the correlation of bond distances in peptide groups detected in ultrahigh-resolution protein structures.

The structural analysis of a deamidated derivative of ribonuclease A, determined at 0.87 A resolution, reveals a highly significant negative correlation between CN and CO bond distances in peptide groups. This trend, i.e. the CO bond lengthens when the CN bond shortens, is also found in seven out of eight protein structures, determined at ultrahigh resolution (<0.95 A). In five of them the linear correlation is statistically significant at the 95% confidence level. The present findings are consistent with the traditional view of amide resonance and, although already found in small peptide structures, they represent a new and important result. In fact, in a protein structure the fine details of the peptide geometry are only marginally affected by the crystal field and they are mostly produced by intramolecular and solvent interactions. The analysis of very high-resolution protein structures can reveal subtle information about local electronic features of proteins which may be critical to folding, function or ligand binding.

The concept of the unfolding region for approaching the mechanisms of enzyme stabilization

Starting from the hypothesis that chemical or genetic modifications as methods of protein stabilization are most successful if the most labile region of the molecule is strengthened, the importance of local structural regions and their modification for thermal stability has been investigated on two enzyme systems. The thermal unfolding of ribonuclease A and its glycosylated form, ribonuclease B, as well as of partially deglycosylated derivatives of ribonuclease B was studied by limited proteolysis. From the analysis of the primary proteolytic cleavage sites and the kinetic stabilities, it can be concluded that the unfolding pathway of ribonuclease A is not changed by the carbohydrate attachment and that the higher stability of ribonuclease B results from the first N-acetylglucosamine residue. On neutral protease from Bacillus stearothermophilus where a surface-located region (residues 56 to 69) is known to be crucial for the thermal stability of the enzyme, the introduction of a disulfide bridge into this region by site-directed mutagenesis resulted in an extreme stabilization of the molecule. Cys residues, which were introduced into different positions of the protein surface, enabled the specific immobilization of the corresponding single mutant enzymes via their SH groups to Activated Thiol-Sepharose. The resulting stabilization effects were highest if the attachment of the enzyme to the carrier was within the residues 56 to 69.

Synthesis of hydrophobic ribonuclease by using reverse micelles. Structural study of AOT reverse micelles containing ribonuclease derivatives

Synthesis of hydrophobic ribonuclease by using reverse micelles. Structural study of AOT reverse micelles containing ribonuclease derivatives

New insolubilized derivatives of ribonuclease and endonuclease for elimination of nucleic acids in single cell protein concentrates.

Two derivatives of pancreatic ribonuclease and endonuclease of Staphylococcus aureus, insolubilized on corn cob, have been used to reduce the percentage of nucleic acids in single cell protein (SCP) concentrates from yeasts. These derivatives are thermostable and active at 45 degrees C. At these temperatures the contamination by bacteria is negligible. The thermostability is remarkable, since the native nuclease is deactivated at above 39 degrees C. The hydrolysis of the nucleic acids in SCP is carried out first with the ribonuclease derivative followed by the endonuclease derivative. The catalytic activity of the insolubilized derivatives is similar to that of the native enzymes in the hydrolysis of RNA but not of DNA. The percentage of nucleic acids is reduced from 5-15 to 0.5%, with a loss of protein of 6%. These percentages are lower than those previously reported.

Crystal structure of two covalent nucleoside derivatives of ribonuclease A

Crystal structures of two forms of ribonuclease A with deoxynucleosides covalently bound to respectively His 12 and His 119 have been solved. One form, T-H12-RNase, has a deoxythymidine bound to N epsilon 2 of His 12, while the other one, U-H119-RNase, has a deoxyuridine bound to N delta 1 of His 119. The two crystal forms are nearly isomorphous, with two molecules in the asymmetric unit. However, the modified ribonucleases differ both in their enzymatic activities and in the conformation of the catalytic site and of the deoxynucleoside-histidine moiety. T-H12-RNase is characterized by complete loss of enzymatic activity; in this form the deoxynucleoside completely blocks the catalytic site and forms intramolecular contacts with residues associated with both the B1 and B2 sites. U-H119-RNase retains 1% of the activity of the unmodified enzyme, and in this form His 119 adopts a different orientation, corresponding to the alternate conformation reported for this residue; the deoxynucleoside-histidine moiety points out of the active site and does not form any contacts with the rest of the protein, thus allowing partial access to the catalytic site. On the basis of these structures, we propose possible mechanisms for the reactions of bromoacetamido nucleosides with ribonuclease A.

Calculation of an OMIT map

A space-group-general computer program to compute an OMIT map using fast Fourier transforms is presented. In this procedure an asymmetric unit of the unit cell is divided into several boxes. For each box a set of phases is calculated by the inverse Fourier transforms of the existing electron density distribution which has been modified so that the electron density values in and around that box is a constant. An OMIT map is then calculated for each box by the Fourier transforms of the observed amplitudes with these phases. The computer program may be used to reduce bias in electron density maps during model building and refinement of macromolecules. The procedure was used to detect error in the atomic model of a derivative of ribonuclease (personal communication by J. Nachman, National Cancer Institute, Frederick, Maryland, USA).

Intermediates in the refolding of ribonuclease at subzero temperatures. 1. Monitoring by nitrotyrosine absorbance.

Derivatives of ribonuclease A in which tyrosines-73, -76, and -115 were nitrated have been synthesized, purified to homogeneity, and characterized by NMR, isoelectric points, absorbance spectra, and catalytic activity. The positions of their reversible thermal unfolding transitions were determined in 35% methanol at pH* 3.0 and 6.0. In the present study the kinetics of the refolding of these nitrotyrosine derivatives were measured at -15 degrees C at pH* 3.0 and 6.0 by using a cryosolvent composed of 35% aqueous methanol. The rates of folding of different regions of the molecule were determined by using the nitrotyrosines as environmentally sensitive probes. Multiphasic kinetics were observed for the refolding of the nitro-Tyr-115, -73, and -76 derivatives. The native environment about Tyr-115 was formed more rapidly than that about Tyr-73 and -76, and the native environment about both these tyrosines was attained much sooner than the native state itself, as judged by other probes. The results indicate that different regions of the molecule attain their native environments at different rates. This observation shows that the folding pathway must involve partially folded intermediate states.

Preliminary crystallographic data for cross-linked (lysine 7-lysine 41)-ribonuclease A

A cross-linked derivative of ribonuclease A, N ε,N ε′ -(2,4-dinitrophenylene-1,5)-(lysine 7-lysine 41)-RNase A, has been crystallized by dialysis against 30% ( v v ) ethanol/water mixtures buffered at high pH. Single crystals belong to the orthorhombic space group P2 12 12 1, a = 37.2 A ̊ , b = 41.2 A ̊ , b = 41.2 A ̊ , with one molecule in the Crystallographic asymmetric unit.

Evidence for interaction of substrate analogs with chicken eggwhite lysozyme after exhaustive reduction of disulfide bonds.

In a study of factors that influence the remaining secondary structure of reduced chicken eggwhite lysozyme, N-acetyl-D-glucosamine (NAG) and N,N'-diacetylchitobiose (di-NAG) were found to alter the circular dichroic (CD) spectrum of the reduced protein and its carboxymethyl derivative (Cml). Thus, negative ellipticities in the far u.v. were greater in the presence of the analogs, with NAG being the more effective. For Cml, curve fitting analysis of the CD data indicated an increased helical content in the presence of NAG by an average of 3% of the chain length, while beta-structure decreased by an equivalent amount. Other compounds structurally related to NAG produced no similar effects on the CD spectrum of Cml, nor were comparable effects of NAG in evidence on the Cm reduced derivatives of ribonuclease, chymotrypsin, wheat germ agglutinin, or alpha-lactalbumin. The effect therefore appears specific between NAG and Cml. Conversion of the tryptophan residue at Position 62 of Cml to the oxindolealanyl derivative prevented these effects of NAG, and this residue may therefore participate in the interaction. During a 4-day incubation at room temperature, the analog preserved the CD spectrum of Cml as well as its concentration. This effect was nearly specific when compared with other Cm reduced proteins and with other carbohydrates. Only one, N-acetyl mannosamine, was effective in preserving the concentration of Cml, but not the CD spectrum. Since D-glucosamine was entirely without effect on either the CD spectrum of Cml or on its change during incubation, the acetyl group appears essential for the NAG-Cml interaction. The specificity between NAG and Cml is tentatively accounted for in terms of interactions with the primary structure, rather than with the remaining secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Ribonuclease inhibitor from human placenta: interaction with derivatives of ribonuclease A.

Ribonuclease inhibitor from human placenta: interaction with derivatives of ribonuclease A.

Endocytes and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo: II. Effect of charge

Experiments presented in this paper suggest that sinusoidal rat liver cells recognize basic on proteins and that this recognition results in endocytosis of the proteins. Evidence for involvement of basic groups was obtained in two ways. Firstly, we changedd the opsitively charged amino groups of the cross-linked ribonuclease molecules to neutral or negative by acetylation or succinylation, respectively. The modified proteins did not contain easily reducible disulfide bonds and they were not very sensitive to endoproteases, suggesting that they were not denatured by the acetylation procedures. Acetylation and succinylation reduced uptake of the injected cross-linked ribonuclease derivatives by liver and spleen and abolsihed their rapid clearance from plasma. In nephrectomized rats about 75% of the polymer, 36% of the acetylated polumer and 32% of the succinylated polumer were endocytosed by liver after 6 h. For the dimer fraction these values were 59%, 23% and 27%, respectively. Autoradiography and subcellular fractionation of liver 30 min post-injection localized the acetylated polumer in the lysosomal/microsomal fraction of sinusoidal liver cessls, probably endothelial cells. Secondly, a positive correlation was found between binding of a number of ribonuclease derivatives of the cation exchanges SP-Sephadex G-25 and the rate of endocytosis by sinusoidal liver cells.

Endocytes and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo: I. Effect of size

Aspects of protein structure determining endocytosis of proteins by sinusoidal rat liver cells in vivo have been studied, using cross-linked or aggregated derivatives of bovine pancreatic ribonuclease A (labelled with 125I) as probes. Ribonuclease was cross-linked by reaction with dimethylsuberimidate, a way of modification that does not change the charge of the protein. Monomer, dimer and polymer fractions were isolated by gel filtration and characterized in respect of size and number of amino groups modified. Maintenance of enzyme activity, stability of disulfide bonds, and lack of susceptibility to endoproteases showed that the cross-linking procedure did not result in gross conformational changes of the ribonuclease molecules. Monomer, dimer and polymer fractions were injected into nephrectomized rats and plasma clearance and uptake in liver and spleen were determined. About 30% of the injected polymer fraction was found in the liver 15 min after injection; for dimer and monomer fractions values of 6% and 2% of the dose were found. Similar differences were found in spleen. Autoradiography, cell isolation, and subcellular fractionation showed that in liver the radioactive proteins were taken up in lysosomes of sinusoidal cells. Similar results were obtained with fractions of aggregated ribonuclease prepared by freeze-drying the protein from 50% acetic acid. Our results demonstrate that the rate of uptake of the ribonuclease derivatives is positively correlated with the size of the molecules. Similarity of the results obtained with cross-linked and aggregated fractions suggests that the numberof ribonuclease ‘subunits’/molecule, rathe than the procedures used to prepare the polymers, determine the rate of uptake by liver and spleen.

Spin-labeled ribonuclease A. Effects of chemical, enzymic, and physical modifications on enzyme conformation

3-SLHis-105-RNase A is an active derivative of ribonuclease A (RNase A) spin-labeled at the 3 position of the imidazole ring of histidine-105. The spin-labeled enzyme has been modified by urea denaturation, reduction, reduction-carboxymethylation, performic acid oxidation, and digestion with proteolytic enzymes in order to monitor changes in the geometry of the protein by changes in the electron paramagnetic resonance (EPR) spectrum of the nitroxide spin-label probe. The results of these experiments indicate that the spin-label attached to histidine-105 of RNase A is sensitive to modifications affecting the conformational integrity of the molecule and to the reconstituting effects of various active-center ligands.

Introduction of a strong binding site for lanthanides at the N-terminus of peptides and ribonuclease A.

A new reagent, phthalyl-4-isothiocyanate, reacts specifically at the N-terminus of a peptide or protein with the production of a phthalic acid 4-thiocarbamyl derivative. The adjacent carboxyl groups of the phthalyl group serve as a strong binding site for lanthanides at pH 5 with a stability constant for praseodymium about 220 times that of a single carboxyl group. Reaction of phthalyl-4-isothiocyanate with α-amino groups of peptides at 25°C in 2H2O at pH meter readings of 8.7 and 7.2 is complete in 15 min and 24 h respectively, without modification of histidine or tyrosine side chains. At the lower pH there is no reaction of the ɛ-amino group of lysine. The phthalic acid 4-thiocarbamyl derivative of ribonuclease A is cyclised and cleaved by the normal Edman degradation in acid to produce the phthalic acid 4-thiohydantoin of lysine. The α-CH proton of lysine-1 in ribonuclease A produces a triplet nuclear magnetic resonance that has been assigned on the basis of its coupling constant, the pH dependence of its chemical shift (pK′ is 7.6) and its disappearance on reaction of ribonuclease A with phthalyl-4-isothiocyanate. The reaction of phthalyl-4-isothiocyanate with a protein allows the introduction of a strong lanthanide binding site specifically at the N-terminus, as a starting point for structural studies using nuclear magnetic resonance spectroscopy.

Analysis of ribonuclease-nucleotide interactions by quantitative affinity chromatography.

Quantitative affinity chromatography on uridine-5'-(Sepharose-4-aminophenylphosphoryl)-2'(3')-phosphate was developed for the study of binding of ribonuclease species to nucleotide ligands. Elution of the native species ribonuclease-A and -S on the afffinity matrix in 0.4 M ammonium acetate, pH 5.2, containing various amounts of the soluble competing ligand 2'-cytidine monophosphate, reveals an inverse response of elution volume to concentration of soluble ligand. This response conforms to behavior expected for the competing binding equilibria enzyme-soluble ligand and enzyme-insoluble ligand. A-NALYSIS OF ELUTION DATA ALLOWS CALCULATION OF KI and KIM, the dissociation constants, respectively, for the soluble and insoluble protein-ligand complexes. The values of these chromatographically derived constants are similar to values of dissocation constants determined in solution by kinetics of inhibition by 2'-cytidine monophosphate and uridine-5'-(j-aminophenylphosphoryl)-2'(3')-phosphate. Successful competitive elution experiments with [p-F-Phe8]semisynthetic ribonuclease-S' and individual elution trials for [4-F-His12]semisynthetic ribonuclease-S' indicate the utility of the quantitative affinity chromatographic technique for determination of ligand binding properties of ribonuclease derivatives, including inactive species. Nonbiospecific aspects of the interaction of ribonuclease with the affinity matrix in ammonium acetate buffers of concentrations 0.1 M and below were noted, delinating limits of conditions allowing the biospecificity needed for ligand-binding analyses by competitive elution. The dependence of ribonuclease competitive elution behavior on the amount of protein eluted also was examined and related to theoretical considerations in the quantitative application of affinity chromatography.

Preparation and properties of three specific active derivatives of ribonuclease A obtained by methylation of methionine residues in 8 M urea

In 8 M urea at low pH, CH3I reacts specifically with the four methionine residues of ribonuclease A, and all four residues react at the same rate. Uon removal of the denaturant, only unmodified ribonuclease and 3 of the 15 possible derivatives modified on methionine refold to regenerate activity. All the enzymatic activity is recored after chromatography on IRC-50 and the four active proteins separate from each other and from the 12 inactive derivatives, which are not eluted from the resin under the conditions used. By the use of 14CH3I, performic acid oxidation, chymotryptic digestion, and separation of the resulting peptides by ion exchange, the active species were determined to be unmodified ribonuclease, CH3Met-29-RNase, CH3Met-79-RNase, and CH3Met-29, CH3Met-79-RNase. these proteins have melting temperatures of 63, 58, 43, and 36 degrees, respectively, at pH 6.3-70. Methylation at methionine-29 or -79 has no effect on enzymatic activity. Conversely, methylation at methionine-13 or -30 prevents refolding to an active conformation at 25 degrees elution from IRC-50. These results are consistent with the positions of the four methionine residues in crystals of ribonuclease A and ribonuclease S as determined by X-ray diffraction.

Assignment of the histidine 12‐threonine 45 hydrogen‐bonded proton in the nmr spectrum of ribonuclease A in H2O

AbstractDescribed herein are proton nmr experiments on chemically modified derivatives of ribonuclease A designed to elucidate the origin of an exchangeable resonance, assigned previously to a histidine ring N proton that titrates between 11 to 13 ppm with a pKa of 6.1 in H2O solution. Histidines 48 and 105, which are distant from the active site, are eliminated as candidates for this resonance from inhibitor binding studies on the enzyme in acetate–water solutions. This exchangeable resonance titrates with modified pKa's and constant area over the above pH range in His‐119‐N1‐carboxymethylated‐RNase A and des‐(121–124)‐RNase A, thus eliminating the imidazole N3 proton in the His 119‐Asp 121 hydrogen bond. In His‐12‐N1‐carboxymethylated‐RNase A, this resonance is also observable, but broadens on raising the pH above 7 and at elevated temperatures above neutrality. It exhibits a pH‐independent chemical shift characteristic of the protonated state of histidine. On the basis of these findings, this exchangeable resonance, designated a, is assigned to the imidazole N1 proton of His 12, which is hydrogen‐bonded to the carbonyl oxygen of Thr 45 in the crystal.

Covalent attachment of proteins to polysaccharide carriers by means of benzoquinone

A new and simple method for the covalent attachment of proteins to polysaccharide carriers using p-benzoquinone is described. Water-insoluble agarose or cross-linked dextran derivatives of ribonuclease, chymotrypsin and serum albumin have been prepared by this method. High amounts of fixed protein are obtained, and the conjugates formed are very stable. Another advantage is that coupling can be obtained over a broad pH range.

The use of three-dimensional direct phase determining methods as applied to the K2PtCl6 derivative of ribonuclease

The calculation of general three-dimensional heavy-atom phases, without assumption of heavy-atom sites, using the tangent formula is investigated. Phases obtained in this way have an average difference of 27 ° as compared with phases calculated for the known heavy-atom constellation in the K2PtCl6 derivative of ribonuclease in space groupP21. Five out of eight of the phase sets produce the correct heavy atom positions by calculation of Δ.E maps.