Picoplatin binding to proteins: X-ray structures and mass spectrometry data on the adducts with lysozyme and ribonuclease A.

The reactivity of the anticancer drug picoplatin (cis-amminedichlorido(2-methylpyridine)platinum(II) complex) with the model proteins hen egg white lysozyme (HEWL) and bovine pancreatic ribonuclease (RNase A) was investigated by electrospray ionisation mass spectrometry (ESI MS) and X-ray crystallography. The data were compared with those previously obtained for the adducts of these proteins with cisplatin, carboplatin and oxaliplatin under the same experimental conditions. ESI-MS data show binding of Pt to both proteins, with fragments retaining the 2-methylpyridine ligand and, possibly, a chloride ion. X-ray crystallography identifies different binding sites on the two proteins, highlighting a different behaviour of picoplatin in the absence or presence of dimethyl sulfoxide (DMSO). Metal-containing fragments bind to HEWL close to the side chains of His15, Asp18, Asp119 and both Lys1 and Glu7, whereas they bind to RNase A on the side chain of His12, Met29, His48, Asp53, Met79, His105 and His119. The data suggest that the presence of DMSO favours the loss of 2-methylpyridine and alters the ability of the Pt compound to bind to the two proteins. With both proteins, picoplatin appears to behave similarly to cisplatin and carboplatin when dissolved in DMSO, whereas it behaves more like oxaliplatin in the absence of the coordinating solvent. This study provides important insights into the pharmacological profile of picoplatin and supports the conclusion that coordinating solvents should not be used to evaluate the biological activities of Pt-based drugs.

Long-timescale atomistic simulations uncover loss-of-function mechanisms of uncharacterized Angiogenin mutants associated with ALS.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive degeneration of motor neurons, resulting in respiratory failure and mortality within 3-5 years. Mutations in the Angiogenin (ANG) cause loss of ribonucleolytic and nuclear translocation activities, contributing to ALS pathogenesis. This study focused on investigating two uncharacterized ANG mutations, T11S and R122H, newly identified in the Project Mine consortium. Using extensive computational analysis, including structural modeling and microsecond-timescale molecular dynamics (MD) simulations, we observed conformational changes in the catalytic residue His114 of ANG induced by T11S and R122H mutations. These alterations impaired ribonucleolytic activity, as inferred through molecular docking and binding free energy calculations. Gibbs free energy landscape and residue-residue interaction network analysis further supported our findings, revealing the energetic states and allosteric pathway from the mutated site to His114. Additionally, we assessed the binding of NCI-65828, an inhibitor of ribonucleolytic activity of ANG, and found reduced effectiveness in binding to T11S and R122H mutants when His114 assumed a non-native conformation. This highlights the crucial role of His114 and its association with ALS. Elucidating the relationship between physical structure and functional dynamics of frequently mutated ANG mutants is essential for understanding ALS pathogenesis and developing more effective therapeutic interventions.

Visualizing ribonuclease digestion of RNA-like polymers produced by hot wet-dry cycles.

Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify that RNA-like polymers can be produced by hot wet-dry cycling of nucleotides. However, it has not been shown that these RNA-like polymers are similar to biological RNA with 3'-5' phosphodiester bonds. In the results described here, RNA-like polymers were generated from 5'-monophosphate nucleosides AMP and UMP. To confirm that the polymers resemble biological RNA, ribonuclease A should catalyze hydrolysis of the 3'-5' phosphodiester bonds between pyrimidine nucleotides to each other or to purine nucleotides, but not purine-purine nucleotide bonds. Here we show AFM images of specific polymers produced by hot wet-dry cycling of AMP, UMP and AMP/UMP (1:1) solutions on mica surfaces, before and after exposure to ribonuclease A. AMP polymers were unaffected by ribonuclease A but UMP polymers disappeared. This indicates that a major fraction of the bonds in the UMP polymers is indeed 3'-5' phosphodiester bonds. Some of the polymers generated from the AMP/UMP mixture also showed clear signs of cleavage. Because ribonuclease A recognizes the ester bonds in the polymers, we show for the first time that these prebiotically produced polymers are in fact similar to biological RNA but are likely to be linked by a mixture of 3'-5' and 2'-5' phosphodiester bonds.

Engineering Human Pancreatic RNase 1 as an Immunotherapeutic Agent for Cancer Therapy Through Computational and Experimental Studies.

Most plant and bacterial toxins are highly immunogenic with non-specific toxic effects. Human ribonucleases are thought to provide a promising basis for reducing the toxic agent's immunogenic properties, which are candidates for cancer therapy. In the cell, the ribonuclease inhibitor (RI) protein binds to the ribonuclease enzyme and forms a tight complex. This study aimed to engineer and provide a gene construct encoding an improved version of Human Pancreatic RNase 1 (HP-RNase 1) to reduce connection to RI and modulate the immunogenic effects of immunotoxins. To further characterize the interaction complex of HP-RNase 1 and RI, we established various in silico and in vitro approaches. These methods allowed us to specifically monitor interactions within native and engineered HP-RNase 1/RI complexes. In silico research involved molecular dynamics (MD) simulations of native and mutant HP-RNase 1 in their free form and when bound to RI. For HP-RNase 1 engineering, we designed five mutations (K8A/N72A/N89A/R92D/E112/A) based on literature studies, as this combination proved effective for the intended investigation. Then, the cDNA encoding HP-RNase 1 was generated by RT-PCR from blood and cloned into the pSYN2 expression vector. Consequently, wild-type and the engineered HP-RNase 1 were over-expressed in E. coli TG1 and purified using an IMAC column directed against a poly-his tag. The protein products were detected by SDS-PAGE and Western blot analysis. HP-RNase 1 catalytic activity, in the presence of various concentrations of RI, demonstrated that the mutated version of the protein is able to escape the ribonuclease inhibitor and target the RNA substrate 2.5 folds more than that of the wild type. From these data, we tend to suggest the engineered recombinant HP-RNase 1 potentially as a new immunotherapeutic agent for application in human cancer therapy.

Sugar osmolyte inhibits and attenuates the fibrillogenesis in RNase A: An in vitro and in silico characterizations.

Mechanisms of protein aggregation are of immense interest in therapeutic biology and neurodegenerative medicine. Biochemical processes within the living cell occur in a highly crowded environment. The phenomenon of macromolecular crowding affects the diffusional and conformational dynamics of proteins and modulates their folding. Macromolecular crowding is reported to cause protein aggregation in some cases, so it is a cause of concern as it leads to a plethora of neurodegenerative disorders and systemic amyloidosis. To divulge the mechanism of aggregation, it is imperative to study aggregation in well-characterized model proteins in the presence of macromolecular crowder. One such protein is ribonuclease A (RNase A), which deciphers neurotoxic function in humans; therefore we decided to explore the amyloid fibrillogenesis of this thermodynamically stable protein. To elucidate the impact of crowder, dextran-70 and its monomer glucose on the aggregation profile of RNase-A various techniques such as Absorbance, Fluorescence, Fourier Transforms Infrared, Dynamic Light Scattering and circular Dichroism spectroscopies along with imaging techniques like Atomic Force Microscopy and Transmission Electron Microscopy were employed. Thermal aggregation and fibrillation were further promoted by dextran-70 while glucose counteracted the effect of the crowding agent in a concentration-dependent manner. This study shows that glucose provides stability to the protein and prevents fibrillation. Intending to combat aggregation, which is the hallmark of numerous late-onset neurological disorders and systemic amyloidosis, this investigation unveils that naturally occurring osmolytes or other co-solutes can be further exploited in novel drug design strategies.

Human RNase 1 can extensively oligomerize through 3D domain swapping thanks to the crucial contribution of its C-terminus.

Human ribonuclease (RNase) 1 and bovine RNase A are the proto-types of the secretory "pancreatic-type" (pt)-RNase super-family. RNase A can oligomerize through the 3D domain swapping (DS) mechanism upon acetic acid (HAc) lyophilisation, producing enzymatically active oligomeric conformers by swapping both N- and C-termini. Also some RNase 1 mutants were found to self-associate through 3D-DS, however forming only N-swapped dimers. Notably, enzymatically active dimers and larger oligomers of wt-RNase 1 were collected here, in higher amount than RNase A, from HAc lyophilisation. In particular, RNase 1 self-associates through the 3D-DS of its N-terminus and, at a higher extent, of the C-terminus. Since RNase 1 is four-residues longer than RNase A, we further analyzed its oligomerization tendency in a mutant lacking the last four residues. The C-terminus role has been investigated also in amphibian onconase (ONC®), a pt-RNase that can form only a N-swapped dimer, since its C-terminus, that is three-residues longer than RNase A, is locked by a disulfide bond. While ONC mutants designed to unlock or cut this constraint were almost unable to dimerize, the RNase 1 mutant self-associated at a higher extent than the wt, suggesting a specific role of the C-terminus in the oligomerization of different RNases. Overall, RNase 1 reaches here the highest ability, among pt-RNases, to extensively self-associate through 3D-DS, paving the way for new investigations on the structural and biological properties of its oligomers.

Cross-Linked Crystals of Dirhodium Tetraacetate/RNase A Adduct Can Be Used as Heterogeneous Catalysts.

Due to their unique coordination structure, dirhodium paddlewheel complexes are of interest in several research fields, like medicinal chemistry, catalysis, etc. Previously, these complexes were conjugated to proteins and peptides for developing artificial metalloenzymes as homogeneous catalysts. Fixation of dirhodium complexes into protein crystals is interesting to develop heterogeneous catalysts. Porous solvent channels present in protein crystals can benefit the activity by increasing the probability of substrate collisions at the catalytic Rh binding sites. Toward this goal, the present work describes the use of bovine pancreatic ribonuclease (RNase A) crystals with a pore size of 4 nm (P3221 space group) for fixing [Rh2(OAc)4] and developing a heterogeneous catalyst to perform reactions in an aqueous medium. The structure of the [Rh2(OAc)4]/RNase A adduct was investigated by X-ray crystallography: the metal complex structure remains unperturbed upon protein binding. Using a number of crystal structures, metal complex accumulation over time, within the RNase A crystals, and structures at variable temperatures were evaluated. We also report the large-scale preparation of microcrystals (∼10-20 µm) of the [Rh2(OAc)4]/RNase A adduct and cross-linking reaction with glutaraldehyde. The catalytic olefin cyclopropanation reaction and self-coupling of diazo compounds by these cross-linked [Rh2(OAc)4]/RNase A crystals were demonstrated. The results of this work reveal that these systems can be used as heterogeneous catalysts to promote reactions in aqueous solution. Overall, our findings demonstrate that the dirhodium paddlewheel complexes can be fixed in porous biomolecule crystals, like those of RNase A, to prepare biohybrid materials for catalytic applications.

Ribonuclease T2 represents a distinct circularly permutated version of the BECR RNases.

Detection of homologous relationships among proteins and understanding their mechanisms of diversification are major topics in the fields of protein science, bioinformatics, and phylogenetics. Recent developments in sequence/profile-based and structural similarity-based methods have greatly facilitated the unification and classification of many protein families into superfamilies or folds, yet many proteins remain unclassified in current protein databases. As one of the three earliest identified RNases in biology, ribonuclease T2, also known as RNase I in Escherichia coli, RNase Rh in fungi, or S-RNase in plant, is thought to be an ancient RNase family due to its widespread distribution and distinct structure. In this study, we present evidence that RNase T2 represents a circularly permutated version of the BECR (Barnase-EndoU-Colicin E5/D-RelE) fold RNases. This subtle relationship cannot be detected by traditional methods such as sequence/profile-based comparisons, structure-similarity searches, and circular permutation detections. However, we were able to identify the structural similarity using rational reconstruction of a theoretical RNase T2 ancestor via a reverse circular permutation process, followed by structural modeling using AlphaFold2, and structural comparisons. This relationship is further supported by the fact that RNase T2 and other typical BECR RNases, namely Colicin D, RNase A, and BrnT, share similar catalytic site configurations, all involving an analogous set of conserved residues on the α0 helix and the ß4 strand of the BECR fold. This study revealed a hidden root of RNase T2 in bacterial toxin systems and demonstrated that reconstruction and modeling of ancestral topology is an effective strategy to identify remote relationship between proteins.

The Non-native Disulfide-Bond-Containing Landscape Orthogonal to the Oxidative Protein-Folding Trajectory: A Necessary Evil?

Oxidative protein folding describes the process by which disulfide-bond-containing proteins mature from their ribosomal, fully reduced and unfolded, origins. Over the past 40 years, a number of exemplar proteins including bovine pancreatic ribonuclease A (RNaseA), bovine pancreatic trypsin inhibitor (BPTI), and hen egg-white lysozyme (HEWL), among others, have provided rich insight into the nature of the intermolecular interactions that drive the formation of the native, biologically active fold. In this Review Article, we revisit the oxidative folding process of RNase A with a focus on reconciling the role of non-native disulfide-bond-containing species that populate the oxidative folding landscape. Toward gaining such an understanding, we project the regeneration pathway onto a Cartesian coordinate system. This helps not only to recognize the magnitude of the seemingly "fruitless", non-native disulfide-bond-containing species that lie orthogonal to the "native-protein-forming" reaction progress but also to reconcile a role for their existence in the regenerative trajectory. Finally, we superimpose the folding funnel onto the regeneration trajectory to draw parallels between oxidative folders and conformational folders (proteins that lack disulfide bonds). The overall objective is to provide the reader with a semi-quantitative description of oxidative protein folding and the barriers to successful regeneration while underscoring a role of seemingly fruitless intermediates.

Structure of angiogenin dimer bound to double-stranded RNA.

Angiogenin is an unusual member of the RNase A family and is of great interest in multiple pathological contexts. Although it has been assigned various regulatory roles, its core catalytic function is that of an RNA endonuclease. However, its catalytic efficiency is comparatively low and this has been linked to a unique C-terminal helix which partially blocks its RNA-binding site. Assuming that binding to its RNA substrate could trigger a conformational rearrangement, much speculation has arisen on the topic of the interaction of angiogenin with RNA. To date, no structural data on angiogenin-RNA interactions have been available. Here, the structure of angiogenin bound to a double-stranded RNA duplex is reported. The RNA does not reach the active site of angiogenin and no structural arrangement of the C-terminal domain is observed. However, angiogenin forms a previously unobserved crystallographic dimer that makes several backbone interactions with the major and minor grooves of the RNA double helix.

Mechanical Stability of Ribonuclease A Heavily Depends on the Redox Environment.

Disulfide bonds are covalent bonds that connect nonlocal fragments of proteins, and they are unique post-translational modifications of proteins. They require the oxidizing environment to be stable, which occurs for example during oxidative stress; however, in a cell the reductive environment is maintained, lowering their stability. Despite many years of research on disulfide bonds, their role in the protein life cycle is not fully understood and seems to strictly depend on a system or process in which they are involved. In this article, coarse-grained UNited RESidue (UNRES), and all-atom Assisted Model Building with Energy Refinement (AMBER) force fields were applied to run a series of steered molecular dynamics (SMD) simulations of one of the most studied, but still not fully understood, proteins─ribonuclease A (RNase A). SMD simulations were performed to study the mechanical stability of RNase A in different oxidative-reductive environments. As disulfide bonds (and any other covalent bonds) cannot break/form in any classical all-atom force field, we applied additional restraints between sulfur atoms of reduced cysteines which were able to mimic the breaking of the disulfide bonds. On the other hand, the coarse-grained UNRES force field enables us to study the breaking/formation of the disulfide bonds and control the reducing/oxidizing environment owing to the presence of the designed distance/orientation-dependent potential. This study reveals that disulfide bonds have a strong influence on the mechanical stability of RNase A only in a highly oxidative environment. However, the local stability of the secondary structure seems to play a major factor in the overall stability of the protein. Both our thermal unfolding and mechanical stretching studies show that the most stable disulfide bond is Cys65-Cys72. The breaking of disulfide bonds Cys26-Cys84 and Cys58-Cys110 is associated with large force peaks. They are structural bridges, which are mostly responsible for stabilizing the RNase A conformation, while the presence of the remaining two bonds (Cys65-Cys72 and Cys40-Cys95) is most likely connected with the enzymatic activity rather than the structural stability of RNase A in the cytoplasm. Our results prove that disulfide bonds are indeed stabilizing fragments of the proteins, but their role is strongly redox environment-dependent.

Hofmeister Effects of Group II Cations as Seen in the Unfolding of Ribonuclease A.

This work studies the effects of alkaline-earth cation addition on the unfolding free energy of a model protein, pancreatic Ribonuclease A (RNase A) by differential scanning calorimetry analysis. RNase A was chosen because: a) it does not specifically bind Mg2+ , Ca2+ and Sr2+ cations and b) maintains its structural integrity throughout a large pH range. We have measured and compared the effects of NaCl, MgCl2 , CaCl2 and SrCl2 addition on the melting point of RNase A. Our results show that even though the addition of group II cations to aqueous solvent reduces the solubility of nonpolar residues (and enhances the hydrophobic effect), their interactions with the amide moieties are strong enough to "salt-them-in" the solvent, thereby causing an overall protein stability reduction. We demonstrate that the amide-cation interactions are a major contributor to the observed "Hofmeister Effects" of group II cations in protein folding. Our analysis suggests that protein folding "Hofmeister Effects" of group II cations, are mostly the aggregate sum of how cation addition simultaneously salts-out hydrophobic moieties by increasing the cavitation free energy, while promoting the salting-in of amide moieties through contact pair formation.

Emerging biological functions of ribonuclease 1 and angiogenin.

Pancreatic-type ribonucleases (ptRNases) are a large family of vertebrate-specific secretory endoribonucleases. These enzymes catalyze the degradation of many RNA substrates and thereby mediate a variety of biological functions. Though the homology of ptRNases has informed biochemical characterization and evolutionary analyses, the understanding of their biological roles is incomplete. Here, we review the functions of two ptRNases: RNase 1 and angiogenin. RNase 1, which is an abundant ptRNase with high catalytic activity, has newly discovered roles in inflammation and blood coagulation. Angiogenin, which promotes neovascularization, is now known to play roles in the progression of cancer and amyotrophic lateral sclerosis, as well as in the cellular stress response. Ongoing work is illuminating the biology of these and other ptRNases.

Reactions with Proteins of Three Novel Anticancer Platinum(II) Complexes Bearing N-Heterocyclic Ligands.

Three novel platinum(II) complexes bearing N-heterocyclic ligands, i.e., Pt2c, Pt-IV and Pt-VIII, were previously prepared and characterized. They manifested promising in vitro anticancer properties associated with non-conventional modes of action. To gain further mechanistic insight, we have explored here the reactions of these Pt compounds with a few model proteins, i.e., hen egg white lysozyme (HEWL), bovine pancreatic ribonuclease (RNase A), horse heart cytochrome c (Cyt-c) and human serum albumin (HSA), primarily through ESI MS analysis. Characteristic and variegate patterns of reactivity were highlighted in the various cases that appear to depend both on the nature of the Pt complex and of the interacting protein. The protein-bound Pt fragments were identified. In the case of the complex Pt2c, the adducts formed upon reaction with HEWL and RNase A were further characterized by solving the respective crystal structures: this allowed us to determine the exact location of the various Pt binding sites. The implications of the obtained results are discussed in relation to the possible mechanisms of action of these innovative anticancer Pt complexes.

Angiogenin and Copper Crossing in Wound Healing.

Angiogenesis plays a key role in the wound healing process, involving the migration, growth, and differentiation of endothelial cells. Angiogenesis is controlled by a strict balance of different factors, and among these, the angiogenin protein plays a relevant role. Angiogenin is a secreted protein member of the ribonuclease superfamily that is taken up by cells and translocated to the nucleus when the process of blood vessel formation has to be promoted. However, the chemical signaling that activates the protein, normally present in the plasma, and the transport pathways through which the protein enters the cell are still largely unclear. Copper is also an angiogenic factor that regulates angiogenin expression and participates in the activation of common signaling pathways. The interaction between angiogenin and copper could be a relevant mechanism in regulating the formation of new blood vessel pathways and paving the way to the development of new drugs for chronic non-healing wounds.

Peptides Derived from Angiogenin Regulate Cellular Copper Uptake.

The angiogenin protein (ANG) is one of the most potent endogenous angiogenic factors. In this work we characterized by means of potentiometric, spectroscopic and voltammetric techniques, the copper complex species formed with peptide fragments derived from the N-terminal domain of the protein, encompassing the sequence 1-17 and having free amino, Ang1-17, or acetylated N-terminus group, AcAng1-17, so to explore the role of amino group in metal binding and cellular copper uptake. The obtained data show that amino group is the main copper anchoring site for Ang1-17. The affinity constant values, metal coordination geometry and complexes redox-potentials strongly depend, for both peptides, on the number of copper equivalents added. Confocal laser scanning microscope analysis on neuroblastoma cells showed that in the presence of one equivalent of copper ion, the free amino Ang1-17 increases cellular copper uptake while the acetylated AcAng1-17 strongly decreases the intracellular metal level. The activity of peptides was also compared to that of the protein normally present in the plasma (wtANG) as well as to the recombinant form (rANG) most commonly used in literature experiments. The two protein isoforms bind copper ions but with a different coordination environment. Confocal laser scanning microscope data showed that the wtANG induces a strong increase in intracellular copper compared to control while the rANG decreases the copper signal inside cells. These data demonstrate the relevance of copper complexes' geometry to modulate peptides' activity and show that wtANG, normally present in the plasma, can affect cellular copper uptake.

An Influence of Modification with Phosphoryl Guanidine Combined with a 2'-O-Methyl or 2'-Fluoro Group on the Small-Interfering-RNA Effect.

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2'-O-methyl (2'-OMe) or PG/2'-fluoro (2'-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.

Dimerization of Human Angiogenin and of Variants Involved in Neurodegenerative Diseases.

Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies.

Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins.

As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein-protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.

Theoretical Studies on the Effect of Isomerized Aspartic Acid Residues on the Three-Dimensional Structures of Bovine Pancreatic Ribonucleases A.

Isomerized aspartic acid (Asp) residues have previously been identified in various aging tissues, and are suspected to contribute to age-related diseases. Asp-residue isomerization occurs nonenzymatically under physiological conditions, resulting in the formation of three types of isomerized Asp (i.e., L-isoAsp, D-Asp, and D-isoAsp) residues. Asp-residue isomerization often accelerates protein aggregation and insolubilization, making structural biology analyses difficult. Recently, Sakaue et al. reported the synthesis of a ribonuclease A (RNase A) in which Asp121 was artificially replaced with different isomerized Asp residues, and experimentally demonstrated that the enzymatic activities of these artificial mutants were completely lost. However, their structural features have not yet been elucidated. In the present study, the three-dimensional (3D) structures of these artificial-mutant RNases A were predicted using molecular dynamics (MD) simulations. The 3D structures of wild-type and artificial-mutant RNases A were converged by 3000-ns MD simulations. Our computational data show that the structures of the active site and the formation frequencies of the appropriate catalytic dyad structures in the artificial-mutant RNases A were quite different from wild-type RNase A. These computational findings may provide an explanation for the experimental data which show that artificial-mutant RNases A lack enzymatic activity. Herein, MD simulations have been used to evaluate the influences of isomerized Asp residues on the 3D structures of proteins.