Apoptin is a nonstructural protein encoded by one of the three open reading frames of the chicken... more Apoptin is a nonstructural protein encoded by one of the three open reading frames of the chicken anemia virus genome. It has attracted a great deal of interest due to its ability to induce apoptosis in multiple transformed and malignant mammalian cell lines without affecting primary and non-transformed cells. However, the use of Apoptin as an antican-cer drug is restricted by its strong tendency to aggregate. A number of methods to overcome this problem have been proposed , including transduction techniques to deliver the Apoptin gene into tumor cells, but all such methods have certain drawbacks. Here we describe that a truncated variant of Apoptin, lacking residues 1 to 43, is a soluble, non-aggregating protein that maintains most of the biological properties of wild-type Apoptin when transfected into cells. We show that the cytotoxic effect of this variant is also present when it is added exogenously to cancer cells, but not to normal cells. In addition to the interest this protein has attracted as a promising therapeutic strategy, it is also an excellent model to study the structural properties of Apoptin and how they relate to its mechanism of action.
Typical antitumor drugs disrupt the flow of biochemical information from DNA to proteins with the... more Typical antitumor drugs disrupt the flow of biochemical information from DNA to proteins with the aim of precluding uncontrolled cell proliferation and inducing cancer cell apoptosis. However, most of the currently used small antitumor drugs are genotoxic because they act over DNA. Pharmaceutical industry is now searching for a new line of cancer chemotherapeutics without genotoxic effects. Ribonucleases (RNases) are small basic proteins, present in all life forms, which belong to this kind of chemotherapeutics. Some of them present with remarkable selective antitumor activity linked to their ability to destroy RNA, a powerful way to control gene expression, leaving DNA unharmed. In the last two decades, the knowledge gained on the cytotoxic mechanism of these RNases has been used to engineer more powerful and selective variants to kill cancer cells. In this chapter, we describe the advances reached in endowing an RNase with antitumor abilities.
Onconase is a ribonuclease that presents both antitumor and antiviral properties linked to its ri... more Onconase is a ribonuclease that presents both antitumor and antiviral properties linked to its ribonucleolytic activity and represents a new class of RNA-damaging drugs. It has reached clinical trials for the treatment of several cancers and human papilloma virus warts. Onconase targets different RNAs in the cell cytosol but Onconase-treated cells present features that are different from a simple arrest of protein synthesis. We have used microarray-derived transcriptional profiling to identify Onconase-regulated genes in two ovarian cancer cell lines (NCI/ADR-RES and OVCAR-8). RT-qPCR analyses have confirmed the microarray findings. We have identified a network of up-regulated genes implicated in different signaling pathways that may explain the cytotoxic effects exerted by Onconase. Among these genes, activating transcription factor 3 (ATF3) plays a central role in the key events triggered by Onconase in treated cancer cells that finally lead to apoptosis. This mechanism, mediated by ATF3, is cell-type independent. Up-regulation of ATF3 may also explain the antiviral properties of this ribonuclease because this factor is involved in halting viral genome replication, keeping virus latency or preventing viral oncogenesis. Finally, Onconase-regulated genes are different from those affected by nuclear-directed ribonucleases.
Keywords: antitumor drug, human pancreatic ribonuclease, metabolism of cancer cells, microarray p... more Keywords: antitumor drug, human pancreatic ribonuclease, metabolism of cancer cells, microarray profiling, tumor cell death ABSTRACT Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted ribonuclease family are not cytotoxic because they are not able to evade the cytosolic ribonuclease inhibitor. We previously engineered the human pancreatic ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21 WAF1/CIP1 induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cells.
RNase A forms 3D domain-swapped oligomers with novel enzymatic and biological activities. We stud... more RNase A forms 3D domain-swapped oligomers with novel enzymatic and biological activities. We study how crowding agents and osmolytes affect the formation and dissociation of RNase A oligomers. The crowding agents Ficoll and dextran were found to enhance oligomer formation, whereas the stabilizers sodium sulfate, glycine and trimethylammonium oxide (TMAO) do not. In contrast, TMAO significantly slows RNase A dimer dissociation, while the effect of Ficoll is small. These results lead us to propose that the mechanisms of oligomer formation and dissociation are different. In the RNase A "C-dimer", the C-terminal β-strand is swapped between two subunits. The loop preceding this β-strand adopts a β-sheet which has been proposed to resemble amyloid structurally. Hydrogen/deuterium (H/D) exchange of the RNase A C-dimer reveal that the H-bonds formed between the swapped C-terminal β-strand and the other subunit are strong. Their rupture may be crucial for C-dimer dissociation. In contrast, H-bonds formed by Asn 113 in the novel β-sheet adopted by the hinge loop in the C-dimer are not strongly protected. Besides the fundamental insights obtained, the results represent a technical advance for obtaining increased oligomer yields and storage lifetimes.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006
This paper gives an overview of the application of high-pressure to study the folding/unfolding p... more This paper gives an overview of the application of high-pressure to study the folding/unfolding processes of proteins using Ribonuclease A as a model protein. A particular focus is the study of pressure-equilibrium unfolding and folding kinetics using variants and the information obtained by comparing these with the wild-type enzyme.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006
Pressure-jump induced relaxation kinetics can be used to study both protein unfolding and refoldi... more Pressure-jump induced relaxation kinetics can be used to study both protein unfolding and refolding. These processes can be initiated by upward and downward pressure-jumps of amplitudes of a few 10 to 100 MPa, with a dead-time on the order of milliseconds. In many cases, the relaxation times can be easily determined when the pressure cell is connected to a spectroscopic detection device, such as a spectrofluorimeter. Adiabatic heating or cooling can be limited by small pressure-jump amplitudes and a special design of the sample cell. Here, we discuss the application of this method to four proteins: 33-kDa and 23-kDa proteins from photo-system II, a variant of the green fluorescent protein, and a fluorescent variant of ribonuclease A. The thermodynamically predicted equivalency of upward and downward pressure-jump induced protein relaxation kinetics for typical two-state folders was observed for the 33-kDa protein, only. In contrast, the three other proteins showed significantly different kinetics for pressure-jumps in opposite directions. These results cannot be explained by sequential reaction schemes. Instead, they are in line with a more complex free energy landscape involving multiple pathways.
In this work we demonstrate that heat and pressure induce only slightly different energetic chang... more In this work we demonstrate that heat and pressure induce only slightly different energetic changes in the unfolded state of RNase A. Using pressure and temperature as denaturants on a significant number of variants, and by determining the free energy of unfolding at different temperatures, we estimated the stability of variants unable to complete the unfolding transition owing to the experimental conditions required for pressure experiments. The overall set of results allowed us to map the contributions to stability of the hydrophobic core residues of RNase A, with the positions most critical for stability being V54, V57, I106 and V108. We also show that the stability differences can be attributed to both hydrophobic interactions and packing density with an equivalent energetic magnitude. The main hydrophobic core of RNase A is tightly packed, as shown by the small-to-large and isosteric substitutions. In addition, we found that large changes in the number of methylene groups have non-additive positive stability interaction energies that are consistent with exquisite tight core packing and rearrangements of van der Waals' interactions in the protein interior, even after drastic deleterious substitutions.
Proteins: Structure, Function, and Bioinformatics, 2009
To investigate the structural origin of decreased pressure and temperature stability, the crystal... more To investigate the structural origin of decreased pressure and temperature stability, the crystal structure of bovine pancreatic ribonuclease A variants V47A, V54A, V57A, I81A, I106A, and V108A was solved at 1.4-2.0 A resolution and compared with the structure of wild-type protein. The introduced mutations had only minor influence on the global structure of ribonuclease A. The structural changes had individual character that depends on the localization of mutated residue, however, they seemed to expand from mutation site to the rest of the structure. Several different parameters have been evaluated to find correlation with decrease of free energy of unfolding DeltaDeltaG(T), and the most significant correlation was found for main cavity volume change. Analysis of the difference distance matrices revealed that the ribonuclease A molecule is organized into five relatively rigid subdomains with individual response to mutation. This behavior consistent with results of unfolding experiments is an intrinsic feature of ribonuclease A that might be surviving remnants of folding intermediates and reflects the dynamic nature of the molecule.
To investigate the characteristics of the postulated carboxy terminal chain-folding initiation si... more To investigate the characteristics of the postulated carboxy terminal chain-folding initiation site in bovine pancreatic ribonuclease A (RNase A) (residues 106-118), important in the early stages of the folding pathway, we have engineered by site-directed mutagenesis a set of 14 predominantly conservative hydrophobic variants of the protein. The stability of each variant has been compared by pressure and temperature-induced unfolding, monitored by fourth derivative UV absorbance spectroscopy. Apparently simple two-state, reversible unfolding transitions are observed, suggesting that the disruption of tertiary structure of each protein at high pressure or temperature is strongly cooperative. Within the limits of the technique, we are unable to detect significant differences between the two processes of denaturation. Both steady-state kinetic parameters for the enzyme reaction and UV CD spectra of each RNase A variant indicate that truncation of hydrophobic side chains in this region has, in general, little or no effect on the native structure and function of the enzyme. Furthermore, the decreases in free energy of unfolding upon pressure and thermal denaturation of all the variants, particularly those modified at residues 106 and 108, suggest that the hydrophobic residues and side chain packing interactions of this region play an important role in maintaining the conformational stability of RNase A. We also demonstrate the potential of Tyr115 replacement by Trp as a non-destabilizing fluorescence probe of conformational changes local to the region.
T hermodynamic and kinetic understanding of structural transformations in proteins is critical to... more T hermodynamic and kinetic understanding of structural transformations in proteins is critical to new developments in medicine and biotechnology. These fields often require the design of mechanism-based modulators of protein function. Researchers increasingly consider these structural changesssuch as folding/unfolding or shuttling between active and inactive statesswithin the energy landscape concept that supposes a high-dimensional, rugged conformational surface. The unevenness, or asperity, of this conformational surface results from energetic barriers and kinetic traps. However, for a large number of protein reactions, such as reversible folding/unfolding, the literature only reports simple two-state transitions, which calls into question the use of a more complex energy landscape model. The question is: are these reactions really that simple, or are we misled by a biased experimental approach? In this Account, we argue in favor of the latter possibility. Indeed, the frequently employed temperature-jump method only allows recording protein structure changes in the heating direction. Under those conditions, it might not be possible to detect other kinetic pathways that could have been taken in the cooling direction.
Protein self-recognition is essential in many biochemical processes and its study is of fundament... more Protein self-recognition is essential in many biochemical processes and its study is of fundamental interest to understand the molecular mechanism of amyloid formation. Ribonuclease A (RNase A) is a monomeric protein that may form several oligomers by 3D domain swapping of its N-terminal alpha-helix, C-terminal beta-strand, or both. RNase A oligomerization is induced by 40% acetic acid, which has been assumed to mildly unfold the protein by detaching the terminal segments and consequently facilitating intersubunit swapping, once the acetic acid is removed by lyophilization and the protein is redissolved in a benign buffer. Using UV difference, near UV circular dichroism, folding kinetics, and multidimensional heteronuclear NMR spectroscopy, the conformation of RNase A in 40% acetic acid and in 8 M urea has been characterized. These studies demonstrate that RNase A is chiefly unfolded in 40% acetic acid; it partially retains the native helices, whereas the beta-sheet is fully denatured and all X-Pro peptide bonds are predominantly in the trans conformation. Refolding occurs via an intermediate, I(N), with non-native X-Pro peptide bonds. I(N) is known to be populated during RNase A refolding following denaturation in concentrated solutions of urea or guanidinium chloride, and we find that urea- or GdmCl-denatured RNase A can oligomerize during refolding. By revealing the importance of a chiefly denaturated state and a refolding intermediate with non-native X-Pro peptide bonds, these findings revise the model for RNase A oligomerization via 3D domain swapping and have general implications for amyloid formation.
RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. Th... more RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. These oligomers show high catalytic activity against double-stranded RNA and striking antitumor actions that are lacking in the monomer. However, the dissociation of these metastable oligomers limits their therapeutic potential. Here, a widely used conjugating agent, 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), has been used to induce the formation of amide bonds between carboxylate and amine groups of different subunits of the RNase A C-dimer. A cross-linked C-dimer which does not dissociate was isolated and was found have augmented enzymatic activity toward double-stranded RNA relative to the unmodified C-dimer. Characterization using chromatography, electrophoresis, mass spectrometry, and NMR spectroscopy revealed that the EDC-treated C-dimer retains its structure and contains one to three novel amide bonds. Moreover, both the EDC-treated C-dimer and EDCtreated RNase A monomer were found to carry an increased number of positive charges (about 6 ( 2 charges per subunit). These additional positive charges are presumably due to adduct formation with EDC, which neutralizes a negatively charged carboxylate group and couples it to a positively charged tertiary amine. The increased net positive charge endowed by EDC adducts likely contributes to the heightened cleavage of double-stranded RNA of the EDC-treated monomer and EDC-treated C-dimer. Further evidence for EDC adduct formation is provided by the reaction of EDC with a dipeptide Ac-Asp-Ala-NH 2 monitored by NMR spectroscopy and mass spectrometry. To determine if EDC adduct formation with proteins is common and how this affects protein net charge, conformation, and activity, four well-characterized proteins, ribonuclease Sa, hen lysozyme, carbonic anhydrase, and hemoglobin, were incubated with EDC and the products were characterized. EDC formed adducts with all these proteins, as judged by mass spectrometry and electrophoresis. Moreover, all suffered conformational changes ranging from slight structural modifications in the case of lysozyme, to denaturation for hemoglobin as measured by NMR spectroscopy and enzyme assays. We conclude that EDC adduct formation with proteins can affect their net charge, conformation, and enzymatic activity.
Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique ... more Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (DH # ), and activation entropy (DS # ) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wildtype protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative DH # of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro 114 isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the b-hairpin comprising Trp 115 . A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel.
Ribonucleases belonging to the pancreatic-type family exhibit a variety of biological activities ... more Ribonucleases belonging to the pancreatic-type family exhibit a variety of biological activities that make them potential candidates as chemotherapeutic agents. Among them are remarkable the selective cytotoxicity against tumor cells, exhibited by onconase, and the bactericidal activity presented by the eosinophil cationic protein (ECP). In the past years, based on what is known about the cytotoxic mechanism of ribonucleases, a lot of work has been performed to switch non-naturally cytotoxic ribonucleases to potent toxins. Most of the efforts have been devoted to the production of ribonucleases endowed with selective cytotoxicity against tumor cells. In the present paper, however, we have used two nonbactericidal ribonucleases, onconase and the human pancreatic ribonuclease, as scaffolds onto which to engineer bactericidal activity. To this end, the main bactericidal determinant described for ECP (YRWR) has been introduced to these proteins either in an internal position or as an extension of the C-terminal end. The ribonucleolytic activity, thermostability, cytotoxicity against eukaryotic cells and the antibacterial activity against Gram-positive and Gram-negative strains have been determined for all the variants produced. The results show that we have endowed both ribonucleases with antibacterial activity against Gram-negative and Gram-positive bacteria. In addition, we show that this activity is, at least in part, dependent on the ribonucleolytic activity of the enzymes. Remarkably, we have developed a human pancreatic ribonuclease variant with de novo acquired selective antibacterial which is not cytotoxic to mammalian cells.
Apoptin is a nonstructural protein encoded by one of the three open reading frames of the chicken... more Apoptin is a nonstructural protein encoded by one of the three open reading frames of the chicken anemia virus genome. It has attracted a great deal of interest due to its ability to induce apoptosis in multiple transformed and malignant mammalian cell lines without affecting primary and non-transformed cells. However, the use of Apoptin as an antican-cer drug is restricted by its strong tendency to aggregate. A number of methods to overcome this problem have been proposed , including transduction techniques to deliver the Apoptin gene into tumor cells, but all such methods have certain drawbacks. Here we describe that a truncated variant of Apoptin, lacking residues 1 to 43, is a soluble, non-aggregating protein that maintains most of the biological properties of wild-type Apoptin when transfected into cells. We show that the cytotoxic effect of this variant is also present when it is added exogenously to cancer cells, but not to normal cells. In addition to the interest this protein has attracted as a promising therapeutic strategy, it is also an excellent model to study the structural properties of Apoptin and how they relate to its mechanism of action.
Typical antitumor drugs disrupt the flow of biochemical information from DNA to proteins with the... more Typical antitumor drugs disrupt the flow of biochemical information from DNA to proteins with the aim of precluding uncontrolled cell proliferation and inducing cancer cell apoptosis. However, most of the currently used small antitumor drugs are genotoxic because they act over DNA. Pharmaceutical industry is now searching for a new line of cancer chemotherapeutics without genotoxic effects. Ribonucleases (RNases) are small basic proteins, present in all life forms, which belong to this kind of chemotherapeutics. Some of them present with remarkable selective antitumor activity linked to their ability to destroy RNA, a powerful way to control gene expression, leaving DNA unharmed. In the last two decades, the knowledge gained on the cytotoxic mechanism of these RNases has been used to engineer more powerful and selective variants to kill cancer cells. In this chapter, we describe the advances reached in endowing an RNase with antitumor abilities.
Onconase is a ribonuclease that presents both antitumor and antiviral properties linked to its ri... more Onconase is a ribonuclease that presents both antitumor and antiviral properties linked to its ribonucleolytic activity and represents a new class of RNA-damaging drugs. It has reached clinical trials for the treatment of several cancers and human papilloma virus warts. Onconase targets different RNAs in the cell cytosol but Onconase-treated cells present features that are different from a simple arrest of protein synthesis. We have used microarray-derived transcriptional profiling to identify Onconase-regulated genes in two ovarian cancer cell lines (NCI/ADR-RES and OVCAR-8). RT-qPCR analyses have confirmed the microarray findings. We have identified a network of up-regulated genes implicated in different signaling pathways that may explain the cytotoxic effects exerted by Onconase. Among these genes, activating transcription factor 3 (ATF3) plays a central role in the key events triggered by Onconase in treated cancer cells that finally lead to apoptosis. This mechanism, mediated by ATF3, is cell-type independent. Up-regulation of ATF3 may also explain the antiviral properties of this ribonuclease because this factor is involved in halting viral genome replication, keeping virus latency or preventing viral oncogenesis. Finally, Onconase-regulated genes are different from those affected by nuclear-directed ribonucleases.
Keywords: antitumor drug, human pancreatic ribonuclease, metabolism of cancer cells, microarray p... more Keywords: antitumor drug, human pancreatic ribonuclease, metabolism of cancer cells, microarray profiling, tumor cell death ABSTRACT Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted ribonuclease family are not cytotoxic because they are not able to evade the cytosolic ribonuclease inhibitor. We previously engineered the human pancreatic ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21 WAF1/CIP1 induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cells.
RNase A forms 3D domain-swapped oligomers with novel enzymatic and biological activities. We stud... more RNase A forms 3D domain-swapped oligomers with novel enzymatic and biological activities. We study how crowding agents and osmolytes affect the formation and dissociation of RNase A oligomers. The crowding agents Ficoll and dextran were found to enhance oligomer formation, whereas the stabilizers sodium sulfate, glycine and trimethylammonium oxide (TMAO) do not. In contrast, TMAO significantly slows RNase A dimer dissociation, while the effect of Ficoll is small. These results lead us to propose that the mechanisms of oligomer formation and dissociation are different. In the RNase A "C-dimer", the C-terminal β-strand is swapped between two subunits. The loop preceding this β-strand adopts a β-sheet which has been proposed to resemble amyloid structurally. Hydrogen/deuterium (H/D) exchange of the RNase A C-dimer reveal that the H-bonds formed between the swapped C-terminal β-strand and the other subunit are strong. Their rupture may be crucial for C-dimer dissociation. In contrast, H-bonds formed by Asn 113 in the novel β-sheet adopted by the hinge loop in the C-dimer are not strongly protected. Besides the fundamental insights obtained, the results represent a technical advance for obtaining increased oligomer yields and storage lifetimes.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006
This paper gives an overview of the application of high-pressure to study the folding/unfolding p... more This paper gives an overview of the application of high-pressure to study the folding/unfolding processes of proteins using Ribonuclease A as a model protein. A particular focus is the study of pressure-equilibrium unfolding and folding kinetics using variants and the information obtained by comparing these with the wild-type enzyme.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006
Pressure-jump induced relaxation kinetics can be used to study both protein unfolding and refoldi... more Pressure-jump induced relaxation kinetics can be used to study both protein unfolding and refolding. These processes can be initiated by upward and downward pressure-jumps of amplitudes of a few 10 to 100 MPa, with a dead-time on the order of milliseconds. In many cases, the relaxation times can be easily determined when the pressure cell is connected to a spectroscopic detection device, such as a spectrofluorimeter. Adiabatic heating or cooling can be limited by small pressure-jump amplitudes and a special design of the sample cell. Here, we discuss the application of this method to four proteins: 33-kDa and 23-kDa proteins from photo-system II, a variant of the green fluorescent protein, and a fluorescent variant of ribonuclease A. The thermodynamically predicted equivalency of upward and downward pressure-jump induced protein relaxation kinetics for typical two-state folders was observed for the 33-kDa protein, only. In contrast, the three other proteins showed significantly different kinetics for pressure-jumps in opposite directions. These results cannot be explained by sequential reaction schemes. Instead, they are in line with a more complex free energy landscape involving multiple pathways.
In this work we demonstrate that heat and pressure induce only slightly different energetic chang... more In this work we demonstrate that heat and pressure induce only slightly different energetic changes in the unfolded state of RNase A. Using pressure and temperature as denaturants on a significant number of variants, and by determining the free energy of unfolding at different temperatures, we estimated the stability of variants unable to complete the unfolding transition owing to the experimental conditions required for pressure experiments. The overall set of results allowed us to map the contributions to stability of the hydrophobic core residues of RNase A, with the positions most critical for stability being V54, V57, I106 and V108. We also show that the stability differences can be attributed to both hydrophobic interactions and packing density with an equivalent energetic magnitude. The main hydrophobic core of RNase A is tightly packed, as shown by the small-to-large and isosteric substitutions. In addition, we found that large changes in the number of methylene groups have non-additive positive stability interaction energies that are consistent with exquisite tight core packing and rearrangements of van der Waals' interactions in the protein interior, even after drastic deleterious substitutions.
Proteins: Structure, Function, and Bioinformatics, 2009
To investigate the structural origin of decreased pressure and temperature stability, the crystal... more To investigate the structural origin of decreased pressure and temperature stability, the crystal structure of bovine pancreatic ribonuclease A variants V47A, V54A, V57A, I81A, I106A, and V108A was solved at 1.4-2.0 A resolution and compared with the structure of wild-type protein. The introduced mutations had only minor influence on the global structure of ribonuclease A. The structural changes had individual character that depends on the localization of mutated residue, however, they seemed to expand from mutation site to the rest of the structure. Several different parameters have been evaluated to find correlation with decrease of free energy of unfolding DeltaDeltaG(T), and the most significant correlation was found for main cavity volume change. Analysis of the difference distance matrices revealed that the ribonuclease A molecule is organized into five relatively rigid subdomains with individual response to mutation. This behavior consistent with results of unfolding experiments is an intrinsic feature of ribonuclease A that might be surviving remnants of folding intermediates and reflects the dynamic nature of the molecule.
To investigate the characteristics of the postulated carboxy terminal chain-folding initiation si... more To investigate the characteristics of the postulated carboxy terminal chain-folding initiation site in bovine pancreatic ribonuclease A (RNase A) (residues 106-118), important in the early stages of the folding pathway, we have engineered by site-directed mutagenesis a set of 14 predominantly conservative hydrophobic variants of the protein. The stability of each variant has been compared by pressure and temperature-induced unfolding, monitored by fourth derivative UV absorbance spectroscopy. Apparently simple two-state, reversible unfolding transitions are observed, suggesting that the disruption of tertiary structure of each protein at high pressure or temperature is strongly cooperative. Within the limits of the technique, we are unable to detect significant differences between the two processes of denaturation. Both steady-state kinetic parameters for the enzyme reaction and UV CD spectra of each RNase A variant indicate that truncation of hydrophobic side chains in this region has, in general, little or no effect on the native structure and function of the enzyme. Furthermore, the decreases in free energy of unfolding upon pressure and thermal denaturation of all the variants, particularly those modified at residues 106 and 108, suggest that the hydrophobic residues and side chain packing interactions of this region play an important role in maintaining the conformational stability of RNase A. We also demonstrate the potential of Tyr115 replacement by Trp as a non-destabilizing fluorescence probe of conformational changes local to the region.
T hermodynamic and kinetic understanding of structural transformations in proteins is critical to... more T hermodynamic and kinetic understanding of structural transformations in proteins is critical to new developments in medicine and biotechnology. These fields often require the design of mechanism-based modulators of protein function. Researchers increasingly consider these structural changesssuch as folding/unfolding or shuttling between active and inactive statesswithin the energy landscape concept that supposes a high-dimensional, rugged conformational surface. The unevenness, or asperity, of this conformational surface results from energetic barriers and kinetic traps. However, for a large number of protein reactions, such as reversible folding/unfolding, the literature only reports simple two-state transitions, which calls into question the use of a more complex energy landscape model. The question is: are these reactions really that simple, or are we misled by a biased experimental approach? In this Account, we argue in favor of the latter possibility. Indeed, the frequently employed temperature-jump method only allows recording protein structure changes in the heating direction. Under those conditions, it might not be possible to detect other kinetic pathways that could have been taken in the cooling direction.
Protein self-recognition is essential in many biochemical processes and its study is of fundament... more Protein self-recognition is essential in many biochemical processes and its study is of fundamental interest to understand the molecular mechanism of amyloid formation. Ribonuclease A (RNase A) is a monomeric protein that may form several oligomers by 3D domain swapping of its N-terminal alpha-helix, C-terminal beta-strand, or both. RNase A oligomerization is induced by 40% acetic acid, which has been assumed to mildly unfold the protein by detaching the terminal segments and consequently facilitating intersubunit swapping, once the acetic acid is removed by lyophilization and the protein is redissolved in a benign buffer. Using UV difference, near UV circular dichroism, folding kinetics, and multidimensional heteronuclear NMR spectroscopy, the conformation of RNase A in 40% acetic acid and in 8 M urea has been characterized. These studies demonstrate that RNase A is chiefly unfolded in 40% acetic acid; it partially retains the native helices, whereas the beta-sheet is fully denatured and all X-Pro peptide bonds are predominantly in the trans conformation. Refolding occurs via an intermediate, I(N), with non-native X-Pro peptide bonds. I(N) is known to be populated during RNase A refolding following denaturation in concentrated solutions of urea or guanidinium chloride, and we find that urea- or GdmCl-denatured RNase A can oligomerize during refolding. By revealing the importance of a chiefly denaturated state and a refolding intermediate with non-native X-Pro peptide bonds, these findings revise the model for RNase A oligomerization via 3D domain swapping and have general implications for amyloid formation.
RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. Th... more RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. These oligomers show high catalytic activity against double-stranded RNA and striking antitumor actions that are lacking in the monomer. However, the dissociation of these metastable oligomers limits their therapeutic potential. Here, a widely used conjugating agent, 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), has been used to induce the formation of amide bonds between carboxylate and amine groups of different subunits of the RNase A C-dimer. A cross-linked C-dimer which does not dissociate was isolated and was found have augmented enzymatic activity toward double-stranded RNA relative to the unmodified C-dimer. Characterization using chromatography, electrophoresis, mass spectrometry, and NMR spectroscopy revealed that the EDC-treated C-dimer retains its structure and contains one to three novel amide bonds. Moreover, both the EDC-treated C-dimer and EDCtreated RNase A monomer were found to carry an increased number of positive charges (about 6 ( 2 charges per subunit). These additional positive charges are presumably due to adduct formation with EDC, which neutralizes a negatively charged carboxylate group and couples it to a positively charged tertiary amine. The increased net positive charge endowed by EDC adducts likely contributes to the heightened cleavage of double-stranded RNA of the EDC-treated monomer and EDC-treated C-dimer. Further evidence for EDC adduct formation is provided by the reaction of EDC with a dipeptide Ac-Asp-Ala-NH 2 monitored by NMR spectroscopy and mass spectrometry. To determine if EDC adduct formation with proteins is common and how this affects protein net charge, conformation, and activity, four well-characterized proteins, ribonuclease Sa, hen lysozyme, carbonic anhydrase, and hemoglobin, were incubated with EDC and the products were characterized. EDC formed adducts with all these proteins, as judged by mass spectrometry and electrophoresis. Moreover, all suffered conformational changes ranging from slight structural modifications in the case of lysozyme, to denaturation for hemoglobin as measured by NMR spectroscopy and enzyme assays. We conclude that EDC adduct formation with proteins can affect their net charge, conformation, and enzymatic activity.
Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique ... more Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (DH # ), and activation entropy (DS # ) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wildtype protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative DH # of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro 114 isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the b-hairpin comprising Trp 115 . A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel.
Ribonucleases belonging to the pancreatic-type family exhibit a variety of biological activities ... more Ribonucleases belonging to the pancreatic-type family exhibit a variety of biological activities that make them potential candidates as chemotherapeutic agents. Among them are remarkable the selective cytotoxicity against tumor cells, exhibited by onconase, and the bactericidal activity presented by the eosinophil cationic protein (ECP). In the past years, based on what is known about the cytotoxic mechanism of ribonucleases, a lot of work has been performed to switch non-naturally cytotoxic ribonucleases to potent toxins. Most of the efforts have been devoted to the production of ribonucleases endowed with selective cytotoxicity against tumor cells. In the present paper, however, we have used two nonbactericidal ribonucleases, onconase and the human pancreatic ribonuclease, as scaffolds onto which to engineer bactericidal activity. To this end, the main bactericidal determinant described for ECP (YRWR) has been introduced to these proteins either in an internal position or as an extension of the C-terminal end. The ribonucleolytic activity, thermostability, cytotoxicity against eukaryotic cells and the antibacterial activity against Gram-positive and Gram-negative strains have been determined for all the variants produced. The results show that we have endowed both ribonucleases with antibacterial activity against Gram-negative and Gram-positive bacteria. In addition, we show that this activity is, at least in part, dependent on the ribonucleolytic activity of the enzymes. Remarkably, we have developed a human pancreatic ribonuclease variant with de novo acquired selective antibacterial which is not cytotoxic to mammalian cells.
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Papers by M. Vilanova