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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 ( P 3 2 21 space group) for fixing [Rh 2 (OAc) 4 ] and developing a heterogeneous catalyst to perform reactions in an aqueous medium. The structure of the [Rh 2 (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 [Rh 2 (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 [Rh 2 (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.

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.

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.

The structure, stability, and enzymatic activity of the adduct formed upon the reaction of the V-picolinato (pic) complex [V IV O(pic) 2 (H 2 O)], with an octahedral geometry and the water ligand in cis to the V═O group, with the bovine pancreatic ribonuclease (RNase A) were studied. While electrospray ionization-mass spectrometry, circular dichroism, and ultraviolet-visible absorption spectroscopy substantiate the interaction between the metal moiety and RNase A, electron paramagnetic resonance (EPR) allows us to determine that a carboxylate group, stemming from Asp or Glu residues, and imidazole nitrogen from His residues are involved in the V binding at acidic and physiological pH, respectively. Crystallographic data demonstrate that the V IV O(pic) 2 moiety coordinates the side chain of Glu111 of RNase A, by substituting the equatorial water molecule at acidic pH. Computational methods confirm that Glu111 is the most affine residue and interacts favorably with the OC -6-23-Δ enantiomer establishing an extended network of hydrogen bonds and van der Waals stabilizations. By increasing the pH around neutrality, with the deprotonation of histidine side chains, the binding of the V complex to His105 and His119 could occur, with that to His105 which should be preferred when compared to that to the catalytically important His119. The binding of the V compound affects the enzymatic activity of RNase A, but it does not alter its overall structure and stability.

Nanocomposite hydrogels containing two-dimensional nanosilicates (NS) have emerged as a new technology for the prolonged delivery of biopharmaceuticals. However, little is known about the physical-chemical properties governing the interaction between NS and proteins and the release profiles of NS-protein complexes in comparison to traditional poly(ethylene glycol) (PEG) hydrogel technologies. To fill this gap in knowledge, we fabricated a nanocomposite hydrogel composed of PEG and laponite and identified simple but effective experimental conditions to obtain sustained protein release, up to 23 times slower as compared to traditional PEG hydrogels, as determined by bulk release experiments and fluorescence correlation spectroscopy. Slowed protein release was attributed to the formation of NS-protein complexes, as NS-protein complex size was inversely correlated with protein diffusivity and release rates. While protein electrostatics, protein concentration, and incubation time were important variables to control protein-NS complex formation, we found that one of the most significant and less appreciated variable to obtain a sustained release of bioactive proteins was the buffer chosen for preparing the initial suspension of NS particles. The buffer was found to control the size of nanoparticles, the absorption potential, morphology, and stiffness of hydrogels. From these studies, we conclude that the PEG-laponite composite fabricated is a promising new platform for sustained delivery of positively charged protein therapeutics.

A protein-based therapy is significantly challenged by the successful delivery of native proteins into the targeted cancer cells. We address this challenge here using an all-sealed divalent aptamer tetrahedral DNA framework (asdTDF) delivery platform, in which the protein drug is encapsulated inside the cavity of the framework stoichiometrically via a reversible chemical bond. The ligase-assisted seal of the nicks results in highly enhanced TDF stability of the against nuclease digestion to effectively protect the therapeutic protein from degradation. In addition, the divalent aptamer sequences incorporated into the framework favor it with a target-specific and efficient delivery capability. Importantly, upon being readily delivered into the targeted cancer cells, endogenous glutathione can trigger the release of the native therapeutic protein from the TDF in a traceless fashion by cleaving the reversible chemical bond, thereby leading to effective apoptosis of the specific cancer cells.

3-Bromo-1,2,4,5-tetrazine has been synthesized in an oxidant- and metal-free method. The synthesis is scalable and relies on inexpensive starting materials. 3-Bromo-1,2,4,5-tetrazine can undergo nucleophilic aromatic substitutions with differently substituted heteroatoms under mild conditions. In particular, its excellent reactivity has been used to attain chemoselective protein labeling. The resulting labeled lysines can react with strained dienophiles to trigger fast click-to-release (CtR) biorthogonal reactions. The characterization of the CtR reaction in physiological conditions and a therapeutically relevant example with the monoclonal antibody Trastuzumab to showcase its application is presented. Finally, 3-bromo-1,2,4,5-tetrazine has been used to achieve site-selective protein labeling through the genetic incorporation of the first unnatural amino acid bearing an unsubstituted 1,2,4,5-tetrazin-3-yl functionality, which can also undergo CtR reactions.

Protein degradation plays various roles in cellular homeostasis and signal transduction. Real-time monitoring of the degradation process not only contributes to the elucidation of relevant biological phenomena but also offers a powerful tool for drug discoveries targeting protein degradation. Fluorescent protein labeling with a protein tag and a synthetic fluorescent probe is a powerful technique that enables the direct visualization of proteins of interest in living cells. Although a variety of protein tags and their labeling probes have been reported, techniques for the visualization of protein degradation in living cells remain limited. In order to overcome this limitation, we herein employed a PYP-tag labeling probe with a fluorescence turn-off switch that enables the imaging of protein degradation. Furthermore, we performed a structure-based design of a PYP-tag to stabilize a complex formed by the probe and the protein tag for long-term live-cell imaging. We successfully applied this technique to live-cell imaging of the degradation process of Regnase-1 in response to immunostimulation.

Quantitative information about protein-ligand interactions is central to drug discovery. To obtain the quintessential reaction dissociation constant, ideally measurements of reactions should be performed without perturbations by molecular labeling or immobilization. The technique of transient induced molecular electrical signal (TIMES) has provided a promising technique to meet such requirements, and its performance in a microfluidic environment further offers the potential for high throughput and reduced consumption of reagents. In this work, we further the development by using integrated TIMES signal (i-TIMES) to greatly enhance the accuracy and reproducibility of the measurement. While the transient response may be of interest, the integrated signal directly measures the total amount of surface charge density resulted from molecules near the surface of electrode. The signals enable quantitative characterization of protein-ligand interactions. We have demonstrated the feasibility of i-TIMES technique using different biomolecules including lysozyme, N , N ', N ″-triacetylchitotriose (TriNAG), aptamer, p -aminobenzamidine (pABA), bovine pancreatic ribonuclease A (RNaseA), and uridine-3'-phosphate (3'UMP). The results show i-TIMES is a simple and accurate technique that can bring tremendous value to drug discovery and research of intermolecular interactions.

Efficient protein delivery into the target cell is highly desirable for protein therapeutics. Current approaches for protein delivery commonly suffer from low-loading protein capacity, poor specificity for target cells, and invisible protein release. Herein, we report a protein@inorganic nanodumpling (ND) system as an intracellular protein delivery platform. Similar to a traditional Chinese food, the dumpling, ND consists of a protein complex "filling" formed by metal-ion-directed self-assembly of protein cargos fused to histidine-rich green fluorescent proteins (H 39 GFPs), which are further encapsulated by an external surface "wrapper" of manganese dioxide (MnO 2 ) via in situ biomineralization. This ND structure allows for a high loading capacity (>63 wt %) for protein cargos with enhanced stability. NDs can be targeted and internalized into cancer cells specifically through folic acid receptors by surface-tailored folic acid. The protein cargo release is in a bistimuli-responsive manner, triggered by an either reductive or acidic intracellular microenvironment. Moreover, the MnO 2 nanowrapper is an efficient fluorescence quencher for inner fused GFPs and also a "switch-on" magnetic resonance imaging (MRI) agent via triggered release of Mn 2+ ions, which enables activatable fluorescence/MRI bimodal imaging of protein release. Finally, the ND is highly potent and specific to deliver functional protein ribonuclease A (RNase A) into cultured target cells and the tumor site in a xenografted mouse model, eliminating the tumor cells with high therapeutic efficacy. Our approach provides a promising alternative to advance protein-based cancer therapeutics.

Characterization of the dynamics of disordered polypeptide chains is required to elucidate the behavior of intrinsically disordered proteins and proteins under non-native states related to the folding process. Here we develop a method using quasielastic neutron scattering, combined with small-angle X-ray scattering and dynamic light scattering, to evaluate segmental motions of proteins as well as diffusion of the entire molecules and local side-chain motions. We apply this method to RNase A under the unfolded and molten-globule (MG) states. The diffusion coefficients arising from the segmental motions are evaluated and found to be different between the unfolded and MG states. The values obtained here are consistent with those obtained using the fluorescence-based techniques. These results demonstrate not only feasibility of this method but also usefulness to characterize the behavior of proteins under various disordered states.

Adenosine and uridine 5'-tetra- and 5'-pentaphosphates were synthesized from an activated tetrametaphosphate ([PPN] 2 [P 4 O 11 ], [PPN] 2 [ 1 ], PPN = bis(triphenylphosphine)iminium) and subsequently tested for inhibition of the enzymatic activity of ribonuclease A (RNase A). Reagent [PPN] 2 [ 1 ] reacts with unprotected uridine and adenosine in the presence of a base under anhydrous conditions to give nucleoside tetrametaphosphates. Ring opening of these intermediates with tetrabutylammonium hydroxide ([TBA][OH]) yields adenosine and uridine tetraphosphates ( p 4 A , p 4 U ) in 92% and 85% yields, respectively, from the starting nucleoside. Treatment of ([PPN] 2 [ 1 ]) with AMP or UMP yields nucleoside-monophosphate tetrametaphosphates ( cp 4 pA, cp 4 pU ) having limited aqueous stability. Ring opening of these ultraphosphates with [TBA][OH] yields p 5 A and p 5 U in 58% and 70% yield from AMP and UMP, respectively. We characterized inorganic and nucleoside-conjugated linear and cyclic oligophosphates as competitive inhibitors of RNase A. Increasing the chain length in both linear and cyclic inorganic oligophosphates resulted in improved binding affinity. Increasing the length of oligophosphates on the 5' position of adenosine beyond three had a deleterious effect on binding. Conversely, uridine nucleotides bearing 5' oligophosphates saw progressive increases in binding with chain length. We solved X-ray cocrystal structures of the highest affinity binders from several classes. The terminal phosphate of p 5 A binds in the P 1 enzymic subsite and forces the oligophosphate to adopt a convoluted conformation, while the oligophosphate of p 5 U binds in several extended conformations, targeting multiple cationic regions of the active-site cleft.

Ribonuclease A (RNase A) is increasingly considered as a biomarker for tumor diagnosis, and it is of great significance to develop an ultrasensitive, cost-effective assay for RNase A detection. Electrochemiluminescence (ECL) technology has distinctive advantages in the development of biosensors for diverse targets. However, most of the ECL biosensors require the complex process of electrode modification, which is laborious and time consuming. In this work, an immobilization-free homogeneous ECL assay was developed for the highly sensitive detection of RNase A activity for the first time. On the basis of the fact that RNase A can specifically hydrolyze RNA at the site of ribonucleotide uracil (rU), a rU-containing chimeric DNA probe is designed and labeled with Ru(bpy) 3 2+ (act as ECL indicator). The chimeric DNA probe hardly diffuses to the surface of negatively charged indium tin oxide (ITO) electrode due to the strong electrostatic repulsion between the negatively charged DNA and ITO electrode, resulting in a weak ECL signal detected. When the RNase A is present, the chimeric DNA probe is hydrolyzed into small fragments, which contains little negative charge and can diffuse easily to the ITO electrode surface due to the decreased electrostatic repulsion. In this case, an enhanced ECL signal can be detected. Under the optimal conditions, there is a linear relationship between the ECL signal and the concentration of RNase A in the range of 0.001-0.10 ng/mL, and the detection limit is 0.2 pg/mL. In addition, the proposed ECL sensing system is also applied to detect the RNase A inhibitor, taking As 3+ as an example. The proposed homogeneous ECL sensing system provides a new approach for the highly sensitive and convenient detection of RNase A as well as other ribonucleases only by redesigning a responding chimeric DNA probe.

The selective and temporal control of protein activity in living cells provides a powerful tool to manipulate cellular function and to develop pro-protein therapeutics (PPT) for targeted therapy. In this work, we reported a facile but general chemical approach to design PPT by modulating protein activity in response to endogenous enzyme of disease cells, and its potential for targeted cancer therapy. We demonstrated that the chemical modification of a protein with quinone propionic acid (QPN), a ligand that could be reduced by tumor-cell-specific NAD(P)H dehydrogenase [quinone] 1 (NQO1), was reversible in the presence of NQO1. Importantly, the QPN-modified cytochrome c (Cyt c-QPN) and ribonuclease A (RNase A-QPN) showed NQO1-regulated protein activity in a highly selective manner. Furthermore, the intracellular delivery of RNase A-QPN using a novel type of lipid-based nanoparticles, and subsequent protein activation by cellular NQO1, selectively inhibit cancer cell growth in vitro and effectively suppress tumor growth in vivo. We believe that our approach increases the number of potentially useful chemical tools for reversibly controlling the structure and function of protein using a disease-cell-specific enzyme, opening opportunities in the study of dynamic biological processes and developing precise protein therapeutics.

Responsive delivery of anticancer proteins into cells is an emerging field in biological therapeutics. Currently, the delivery of proteins is highly compromised by multiple successive physiological barriers that reduce the therapeutic efficacy. Hence, there is a need to design a robust and sustainable nanocarrier to provide suitable protection of proteins and overcome the physiological barriers for better cellular accumulation. In this work, polyethylenimine (PEI) cross-linked by oxaliplatin(IV) prodrug (oxliPt(IV)) was used to fabricate a redox-responsive nanocomplex (PEI-oxliPt(IV)@RNBC/GOD) for the delivery of a reactive oxygen species-cleavable, reversibly caged RNase A protein (i.e., RNase A nitrophenylboronic conjugate, RNBC) and glucose oxidase (GOD) in order to realize efficient cancer treatment. The generation of hydrogen peroxide by GOD can uncage and restore the enzymatic activity of RNBC. On account of the responsiveness of the nanocomplex to highly reducing cellular environment, it would dissociate and release the protein and active oxaliplatin drug, causing cell death by both catalyzing RNA degradation and inhibiting DNA synthesis. As assessed by the RNA degradation assay, the activity of the encapsulated RNBC was recovered by the catalytic production of hydrogen peroxide from GOD and glucose substrate overexpressed in cancer cells. Monitoring of the changes in nanoparticle size confirmed that the nanocomplex could dissociate in the reducing environment, with the release of active oxaliplatin drug and protein. Confocal laser scanning microscopy (CLSM) and flow cytometry analysis revealed highly efficient accumulation of the nanocomplex as compared to free native proteins. In vitro cytotoxicity experiments using 4T1 cancer cells showed ∼80% cell killing efficacy, with highly efficient apoptosis induction. Assisted by the cationic polymeric carrier, it was evident from CLSM images that intracellular delivery of the therapeutic protein significantly depleted the RNA level. Thus, this work provides a promising platform for the delivery of therapeutic proteins and chemotherapeutic drugs for efficient cancer treatment.

Delivery of proteins to carry out desired biological functions is a direct approach for disease treatment. However, protein therapy is still facing challenges due to low delivery efficiency, poor targeting during trafficking, insufficient therapeutic efficacy, and possible toxicity induced by carriers. Here, we present a novel delivery platform based on DNA origami nanostructure that enables tumor cell transportation of active proteins for cancer therapy. In our design, cytotoxic protein ribonuclease (RNase) A molecules are organized on the rectangular DNA origami nanosheets, which work as nanovehicles to deliver RNase A molecules into the cytoplasm and execute their cell-killing function inside the tumor cells. Cancer cell-targeting aptamers are also integrated onto the DNA origami-based nanoplatform to enhance its targeting effect. This DNA origami-protein coassembling strategy can be further developed to transport other functional proteins and therapeutic components simultaneously for synergistic effects and be adapted for integrated diagnostics and therapeutics.

Nucleic acid nanostructures have attracted significant interest as potential therapeutic and diagnostic platforms due to their intrinsic biocompatibility and biodegradability, structural and functional diversity, and compatibility with various chemistries for modification and stabilization. Among the fabrication approaches for such structures, the rolling circle techniques have emerged as particularly promising, producing morphologically round, flower-shaped nucleic acid particles: typically hybrid composites of long nucleic acid strands and inorganic magnesium pyrophosphate (Mg 2 PPi). These constructs are known to form via anisotropic nucleic acid-driven crystallization in a sequence-independent manner, rendering monodisperse and densely packed RNA or DNA-inorganic composites. However, it still remains to fully explore how flexible polymer-like RNA or DNA strands (acting as biomolecular additives) mediate the crystallization process of Mg 2 PPi and affect the structure and properties of the product crystals. To address this, we closely examined nanoscale details to mesoscopic features of Mg 2 PPi/DNA hybrid composites fabricated by two approaches, namely rolling circle amplification (RCA)-based in situ synthesis and long synthetic DNA-mediated crystallization. Similar to the DNA constructs fabricated by RCA, the rapid crystallization of Mg 2 PPi was retarded on a short-range order when we precipitated the crystals in the presence of presynthesized long DNA, which resulted in effective incorporation of biomolecular additives such as DNA and enzymes. These findings further provide a more feasible way to encapsulate bioactive enzymes within DNA constructs compared to in situ RCA-mediated synthesis, i.e., by not only protecting them from possible denaturation under the reaction conditions but also preventing nonselective association of proteins arising from the RCA reaction mixtures.

The ability to design surfaces with reversible, high-affinity protein binding sites represents a significant step forward in the advancement of analytical methods for diverse biochemical and biomedical applications. Herein, we report a dynamic supramolecular strategy to directly assemble proteins on surfaces based on multivalent host-guest interactions. The host-guest interactions are achieved by one-step nanofabrication of a well-oriented β-cyclodextrin host-derived self-assembled monolayer on gold (β-CD-SAM) that forms specific inclusion complexes with hydrophobic amino acids located on the surface of the protein. Cytochrome c, insulin, α-chymotrypsin, and RNase A are used as model guest proteins. Surface plasmon resonance and static time-of-flight secondary ion mass spectrometry studies demonstrate that all four proteins interact with the β-CD-SAM in a specific manner via the hydrophobic amino acids on the surface of the protein. The β-CD-SAMs bind the proteins with high nanomolar to single-digit micromolar dissociation constants ( K D ). Importantly, while the proteins can be captured with high affinity, their release from the surface can be achieved under very mild conditions. Our results expose the great advantages of using a supramolecular approach for controlling protein immobilization, in which the strategy described herein provides unprecedented opportunities to create advanced bioanalytic and biosensor technologies.

Ribonuclease 1 (RNase 1) is the most prevalent human homologue of the archetypal enzyme RNase A. RNase 1 contains sequons for N-linked glycosylation at Asn34, Asn76, and Asn88 and is N-glycosylated at all three sites in vivo. The effect of N-glycosylation on the structure and function of RNase 1 is unknown. By using an engineered strain of the yeast Pichia pastoris, we installed a heptasaccharide (Man 5 GlcNAc 2 ) on the side chain of Asn34, Asn76, and Asn88 to produce the authentic triglycosylated form of human RNase 1. As a glutamine residue is not a substrate for cellular oligosaccharyltransferase, we used strategic asparagine-to-glutamine substitutions to produce the three diglycosylated and three monoglycosylated forms of RNase 1. We found that the N-glycosylation of RNase 1 at any position attenuates its catalytic activity but enhances both its thermostability and its resistance to proteolysis. N-Glycosylation at Asn34 generates the most active and stable glycoforms, in accord with its sequon being highly conserved among vertebrate species. These data provide new insight on the biological role of the N-glycosylation of a human secretory enzyme.

Protein-based drugs are a promising class of therapeutics, but poor membrane permeability typically limits their application to extracellular receptors. Delivery strategies that can transport functional proteins to reach intracellular targets are needed, but with many current approaches, biomolecules become entrapped in the endosomes. This greatly reduces the effective concentrations of therapeutic agents at the target sites. Herein, we report a bioconjugation-based approach for intracellular protein delivery by site-selectively attaching amphiphilic polymers to the N-terminal positions of proteins using 2-pyridinecarboxaldehyde groups. The reaction is simple and features mild, aqueous conditions with no required genetic engineering of the proteins. Imaging studies demonstrate that the polymer-protein conjugates are successfully delivered into the cytosol of various cancer cell lines, likely through a membrane fusion mechanism. When conjugated to the delivery polymers, the activity of modified RNase A was retained and notably promoted cytotoxicity in cancer cells upon delivery to the cytosol. This work therefore provides a promising platform for protein-based material delivery for therapeutic applications.

Recent advances in the analysis of proteins have increased the demand for more efficient techniques to separate intact proteins. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquefied CO 2 to conventional liquid mobile phases. The addition of liquefied CO 2 increases diffusivity and decreases viscosity, which inherently leads to a more efficient separation. Herein, EFLC is applied to hydrophobic interaction chromatography (HIC) stationary phases for the first time to study the impact of liquefied CO 2 to the chromatographic behavior of proteins. The effects of liquefied CO 2 on chromatographic properties, charge state distributions (CSDs), and ionization efficiencies were evaluated. EFLC offered improved chromatographic performance compared to conventional liquid chromatography (LC) methods including a shorter analysis time, better peak shapes, and higher plate numbers. The addition of liquefied CO 2 to the mobile phase provided an electrospray ionization (ESI)-friendly and "supercharging" reagent without sacrificing chromatographic performance, which can be used to improve peptide and protein identification in large-scale application.

The mechanisms of electrostatically driven adsorption of proteins on charged surfaces are studied with a new theoretical framework. The acid-base behavior, charge distribution, and electrostatic contributions to the thermodynamic properties of the proteins are modeled in the presence of a charged surface. The method is validated against experimental titration curves and apparent p K a s. The theory predicts that electrostatic interactions favor the adsorption of proteins at their isoelectric points on charged surfaces despite the fact that the protein has no net charge in solution. Two known mechanisms explain adsorption under these conditions: (i) charge regulation (the charge of the protein changes due to the presence of the surface) and (ii) charge patches (the protein orients to place charged amino acids near opposite surface charges). This work shows that both mechanisms contribute to adsorption at low ionic strengths, whereas only the charge-patch mechanism operates at high ionic strength. Interestingly, the contribution of charge regulation is insensitive to protein orientation under all conditions, which validates the use of constant-charge simulations to determine the most stable orientation of adsorbed proteins. The present study also shows that the charged surface can induce large shifts in the apparent p K a s of individual amino acids in adsorbed proteins. Our conclusions are valid for all proteins studied in this work (lysozyme, α-amylase, ribonuclease A, and β-lactoglobulin), as well as for proteins that are not isoelectric but have instead a net charge in solution of the same sign as the surface charge, i.e. the problem of protein adsorption on the "wrong side" of the isoelectric point.

Highly reactive intermediates of the Maillard reaction, such as glycolaldehyde and glyoxal, are precursors in the modification and cross-linking of proteins. Therefore, we investigated ribonuclease A modified by glycolaldehyde and glyoxal, separately. For the first time, various protein species derived by these aldehydes were successfully separated by ion-exchange chromatography and gel permeation chromatography. Highly cross-linked ribonuclease A was obtained in glycolaldehyde incubations. In contrast, glyoxal predominantly led to modified monomeric protein species. These results were verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing. Quantitation of mono- and bivalent protein modifications of the isolated protein species led to a positive correlation between the degree of protein modification and the change of the isoelectric point and molecular weight, respectively. Glycolaldehyde is easily oxidized to glyoxal. However, significantly lower levels of bivalent glyoxal modifications were detected in glycolaldehyde versus glyoxal incubations (glyoxal-lysine dimer, 1.58 ± 0.02 versus 2.86 ± 0.04 mmol/mol of phenylalanine; glyoxal-lysine amide, 2.7 ± 0.1 versus 5.6 ± 0.1 mmol/mol of phenylalanine). In addition, a novel glycolaldehyde-specific lysine-lysine cross-link was identified and putatively assigned as 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentyl-amino)pyridinium salt.

The CHARMM36 carbohydrate parameter set did not adequately reproduce experimental thermodynamic data of carbohydrate interactions with water or proteins or carbohydrate self-association; thus, a new nonbonded parameter set for carbohydrates was developed. The parameters were developed to reproduce experimental Kirkwood-Buff integral values, defined by the Kirkwood-Buff theory of solutions, and applied to simulations of glycerol, sorbitol, glucose, sucrose, and trehalose. Compared to the CHARMM36 carbohydrate parameters, these new Kirkwood-Buff-based parameters reproduced accurately carbohydrate self-association and the trend of activity coefficient derivative changes with concentration. When using these parameters, preferential interaction coefficients calculated from simulations of these carbohydrates and the proteins lysozyme, bovine serum albumin, α-chymotrypsinogen A, and RNase A agreed well with the experimental data, whereas use of the CHARMM36 parameters indicated preferential inclusion of carbohydrates, in disagreement with the experiment. Thus, calculating preferential interaction coefficients from simulations requires using a force field that accurately reproduces trends in the thermodynamic properties of binary excipient-water solutions, and in particular the trend in the activity coefficient derivative. Finally, the carbohydrate-protein simulations using the new parameters indicated that the carbohydrate size was a major factor in the distribution of different carbohydrates around a protein surface.

Understanding how oxidatively damaged RNA interacts with ribonucleases is important because of its proposed role in the development and progression of disease. Thus, understanding structural aspects of RNA containing lesions generated under oxidative stress, as well as its interactions with other biopolymers, is fundamental. We explored the reactivity of RNase A, RNase T 1 , and RNase H toward oligonucleotides of RNA containing 8-oxo-7,8-dihydroguanosine (8oxoG). This is the first example that addresses this relationship and will be useful for understanding (1) how these RNases can be used to characterize the structural impact that this lesion has on RNA and (2) how oxidatively modified RNA may be handled intracellularly. 8-OxoG was incorporated into 10-16-mers of RNA, and its reactivity with each ribonuclease was assessed via electrophoretic analyses, circular dichroism, and the use of other C8-purine-modified analogues (8-bromoguanosine, 8-methoxyguanosine, and 8-oxoadenosine). RNase T 1 does not recognize sites containing 8-oxoG, while RNase A recognizes and cleaves RNA at positions containing this lesion while differentiating if it is involved in H-bonding. The selectivity of RNase A followed the order C > 8-oxoG ≈ U. In addition, isothermal titration calorimetry showed that an 8-oxoG-C3'-methylphosphate derivative can inhibit RNase A activity. Cleavage patterns obtained from RNase H displayed changes in reactivity in a sequence- and concentration-dependent manner and displayed recognition at sites containing the modification in some cases. These data will aid in understanding how this modification affects reactivity with ribonucleases and will enable the characterization of global and local structural changes in oxidatively damaged RNA.

The understanding of fast folding dynamics of single α-helices comes mostly from studies on rationally designed peptides displaying sequences with high helical propensity. The folding/unfolding dynamics and energetics of α-helix conformations in naturally occurring peptides remains largely unexplored. Here we report the study of a protein fragment analogue of the C-peptide from bovine pancreatic ribonuclease-A, RN80, a 13-amino acid residue peptide that adopts a highly populated helical conformation in aqueous solution. 1 H NMR and CD structural studies of RN80 showed that α-helix formation displays a pH-dependent bell-shaped curve, with a maximum near pH 5, and a large decrease in helical content in alkaline pH. The main forces stabilizing this short α-helix were identified as a salt bridge formed between Glu-2 and Arg-10 and the cation-π interaction involving Tyr-8 and His-12. Thus, deprotonation of Glu-2 or protonation of His-12 are essential for the RN80 α-helix stability. In the present study, RN80 folding and unfolding were triggered by laser-induced pH jumps and detected by time-resolved photoacoustic calorimetry (PAC). The photoacid proton release, amino acid residue protonation, and unfolding/folding events occur at different time scales and were clearly distinguished using time-resolved PAC. The partial unfolding of the RN80 α-helix, due to protonation of Glu-2 and consequent breaking of the stabilizing salt bridge between Glu-2 and Arg-10, is characterized by a concentration-independent volume expansion in the sub-microsecond time range (0.8 mL mol -1 , 369 ns). This small volume expansion reports the cost of peptide backbone rehydration upon disruption of a solvent-exposed salt bridge, as well as backbone intrinsic expansion. On the other hand, RN80 α-helix folding triggered by His-12 protonation and subsequent formation of a cation-π interaction leads to a microsecond volume contraction (-6.0 mL mol -1 , ∼1.7 μs). The essential role of two discrete side chain interactions, a salt bridge, and in particular a single cation-π interaction in the folding dynamics of a naturally occurring α-helix peptide is uniquely revealed by these data.

This article describes the synthesis and characterization of novel cationic five-coordinate Pt(II) complexes containing nitrogen sugar-based ligands. The cytotoxicity of the complexes was evaluated on different cell lines with the expectation that both the coordinative saturation and the sugar moiety cooperate to enhance their biological activity. In fact, the complexes resulted to be more active than cisplatin but still with little selectivity. They activate the apoptosis pathway. Binding of representative compounds with DNA was studied by ethidium bromide displacement assay and circular dichroism. Binding to model proteins was also investigated; the X-ray structure of the adduct formed in the reaction between a representative compound and the model protein bovine pancreatic ribonuclease was obtained. The structure discloses an unprecedented interaction between a five-coordinate Pt(II) moiety and a His side chain.

When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two-state transitions during chemical denaturation. The extent of stability exerted by Glu is higher for RNase A at higher temperature, whereas it provides more stability for α-LA at lower temperature. Thus, the experiments indicate that Glu induces a thermal equilibrium intermediate and increases the thermodynamic stability of proteins irrespective of their surface charges. The extent of stability varies between the proteins in a temperature-dependent manner.

Selenoglutathione (GSeH) is a selenium analogue of naturally abundant glutathione (GSH). In this study, this water-soluble small tripeptide was synthesized in a high yield (up to 98%) as an oxidized diselenide form, i.e., GSeSeG (1), by liquid-phase peptide synthesis (LPPS). Obtained 1 was applied to the investigation of the glutathione peroxidase (GPx)-like catalytic cycle. The important intermediates, i.e., GSe - and GSeSG, besides GSeO 2 H were characterized by 77 Se NMR spectroscopy. Thiol exchange of GSeSG with various thiols, such as cysteine and dithiothreitol, was found to promote the conversion to GSe - significantly. In addition, disproportionation of GSeSR to 1 and RSSR, which would be initiated by heterolytic cleavage of the Se-S bond and catalyzed by the generated selenolate, was observed. On the basis of these redox behaviors, it was proposed that the heterolytic cleavage of the Se-S bond can be facilitated by the interaction between the Se atom and an amino or aromatic group, which is present at the GPx active site. On the other hand, when a catalytic amount of 1 was reacted with scrambled 4S species of RNase A in the presence of NADPH and glutathione reductase, native protein was efficiently regenerated, suggesting a potential use of 1 to repair misfolded proteins through reduction of the non-native SS bonds.

Reversible regulation of enzyme activity by chemical and physical stimuli is often achieved by incorporating stimuli-responsive domains in the enzyme of interest. However, this method is suitable for a limited number of enzymes with well-defined structural and conformational changes. In this study, we present a method to encapsulate enzymes in a DNA cage that could transform its conformation depending on the pH, allowing reversible control of the accessibility of the enzyme to the surrounding environment. This enabled us to regulate various properties of the enzyme, such as its resistance to protease-dependent degradation, binding affinity to the corresponding antibody, and most importantly, enzyme activity. Considering that the size and pH responsiveness of the DNA cage can be easily adjusted by the DNA length and sequence, our method provides a broad-impact platform for controlling enzyme functions without modifying the enzyme of interest.

Here, we report novel lipo-oligoaminoamide nanoformulations for targeted intracellular protein delivery. Formulations are generated by first bioreversibly conjugating a sequence-defined amphiphilic lipo-oligomer 728 to the cargo protein via disulfide bonds, followed by formulation of the formed 728-SS-protein conjugate with different helper lipids in various compositions. The triblock oligoaminoamide 728 contains cysteines for reversible covalent protein conjugation and cross-link-stabilization of formed nanoparticles, polyethylene glycol (PEG) for shielding, and providing a hydrophilic domain, eight cationizable succinoyl tetraethylene pentamine (Stp) repeats for endosomal buffering and escape into the cytosol, and a tetra-oleic acid block for hydrophobic stabilization. The added helper lipids are supposed to enhance serum stability of the nanoparticles and provide targeting by lipid-anchored folic acid (FA)-PEG. The optimized protein nanoparticles, including 728, DOPS, cholesterol, DMPE-PEG2000, and the FA-PEG conjugated lipid 1042, presented a high colloidal stability without significant size increase in 72 h. Using cytotoxic ribonuclease A (RNase A) as cargo protein, FA-728-DOPS-DMPE-RNase A nanoformulation could be identified with highest potency of targeted RNase A-mediated folate-receptor-positive KB carcinoma cell killing among all tested formulations, resulting in 85% KB cell killing at a low concentration of 2 μM. These approximately 50 nm sized nanoparticles induced superior 70% KB cell killing even in the presence of 20% serum. Efficient targeted cytosolic delivery by coformulation with helper lipids was also demonstrated by FA-728-DOPS-DMPE-nlsEGFP nanoformulation using enhanced green fluorescent protein (EGFP) as cargo. Furthermore, partial nlsEGFP was imported into the nuclei of KB cells, validating effective endosomal escape, and following nuclear transport mediated by nuclear localization signal on nlsEGFP. As demonstrated, the screening and optimization of nanoformulations with helper lipids and coformulation agents is considered to be an important and rational next step in the development of intracellular biopharmaceuticals, following initial protein conjugate synthesis.

pH is a key parameter for technological and biological processes, intimately related to biomolecular charge. As such, it controls biomolecular conformation and intermolecular interactions, for example, protein/RNA stability and folding, enzyme activity, regulation through conformational switches, protein-polyelectrolyte association, and protein-RNA interactions. pH also plays an important role in technological systems in food, brewing, pharma, bioseparations, and biomaterials in general. Predicting the structure of large proteins and complexes remains a great challenge experimentally, industrially, and theoretically, despite the variety of numerical schemes available ranging from Poisson-Boltzmann approaches to explicit solvent based methods. In this work we benchmark a fast proton titration scheme against experiment and several theoretical methods on the following set of representative proteins: [HP36, BBL, HEWL (triclinic and orthorhombic), RNase, SNASE (V66K/WT, V66K/PHS, V66K/Δ+PHS, L38D/Δ+PHS, L38E/Δ+PHS, L38K/Δ+PHS), ALAC, and OMTKY3]; routinely used in similar tests due to the diversity of their structural features. Our scheme is rooted in the classical Tanford-Kirkwood model of impenetrable spheres, where salt is treated at the Debye-Hückel level. Treating salt implicitly dramatically reduces the computation time, thereby circumventing sampling difficulties faced by other numerical schemes. In comparison with experimental measurements, our calculated pK a values have the average, maximum absolute, and root-mean-square deviations of 0.4-0.9, 1.0-5.2, and 0.5-1.2 pH units, respectively. These values are within the ranges commonly observed in theoretical models. They are also in the large majority of the cases studied here more accurate than the NULL model. For BBL, ALAC, and OMTKY3, the predicted pK a are closer to experimental results than other analyzed theoretical data. Despite the intrinsic approximations of the fast titration scheme, its robustness and ability to properly describe the main system physics is confirmed.

The specific transport of bioactive proteins into designated target cells is an interesting and challenging perspective for the generation of innovative biopharmaceuticals. Natural protein cytotoxins perform this task with outstanding efficacy. They enter cells with receptor-targeted specificity, respond to changing intracellular microenvironments, and by various mechanisms translocate their cytotoxic protein subunit into the cytosol. Here we imitate this toxin-based delivery strategy in an artificial setting, by bioreversible conjugation of a cytotoxic cargo protein (RNase A) with receptor-targeting PEG-folate and the pH-specific endosomolytic peptide INF7 as synthetic delivery domains. Covalent modification of the cargo protein was achieved using the pH-labile AzMMMan linker and copper-free click chemistry with DBCO-modified delivery modules. This linkage is supposed to enable traceless intracellular release of the RNase A after exposure to the endosomal weakly acidic environment. Delivery of RNase A via this polycation-free delivery strategy resulted in high cytotoxicity against receptor-positive KB tumor cells only when both PEG-folate and INF7 were attached.

Translational diffusion is the most fundamental form of transport in chemical and biological systems. The diffusion coefficient is highly sensitive to changes in the size of the diffusing species; hence, it provides important information on the variety of macromolecular processes, such as self-assembly or folding-unfolding. Here, we investigate the behavior of the diffusion coefficient of a macromolecule in the vicinity of heat-induced transition from folded to unfolded state. We derive the equation that describes the diffusion coefficient of the macromolecule in the vicinity of the transition and use it to fit the experimental data from pulsed-field-gradient nuclear magnetic resonance (PFG NMR) experiments acquired for two globular proteins, lysozyme and RNase A, undergoing temperature-induced unfolding. A very good qualitative agreement between the theoretically derived diffusion coefficient and experimental data is observed.

We present a benchmark study of spectral simulation protocols for the amide I band of proteins. The amide I band is widely used in infrared spectroscopy of proteins due to the large signal intensity, high sensitivity to hydrogen bonding, and secondary structural motifs. This band has, thus, proven valuable in many studies of protein structure-function relationships. We benchmark spectral simulation protocols using two common force fields in combination with several electrostatic mappings and coupling models. The results are validated against experimental linear absorption and two-dimensional infrared spectroscopy for three well-studied proteins. We find two-dimensional infrared spectroscopy to be much more sensitive to the simulation protocol than linear absorption and report on the best simulation protocols. The findings demonstrate that there is still room for ideas to improve the existing models for the amide I band of proteins.

Intracellularly-acting therapeutic proteins are considered promising alternatives for the treatment of various diseases. Major limitations of their application are low efficiency of intracellular delivery and possible reduction of protein activity during derivatization. Herein, we report pH-sensitive covalent modification of proteins with a histidine-rich cationic oligomer (689) for efficient intracellular transduction and traceless release of functional proteins. Enhanced Green fluorescent protein (EGFP), as model for the visualization of protein transduction, and RNase A, as therapeutic protein with antitumoral effect, were modified with the pH-sensitive bifunctional AzMMMan linker and varying amounts of cationic oligomer. The modification degree showed impact on the internalization and cellular distribution of EGFP as well as the biological effect of RNase A conjugates, which mediated considerable toxicity against cancer cells at optimal ratio. The presented conjugates demonstrate their qualification to achieve efficient intracellular delivery and controlled release without protein inactivation and potential prospective applications in protein-based therapies.

The contribution of noncovalent interactions to the stability of naturally occurring peptides and proteins has been generally acknowledged, though how these can be rationally manipulated to improve the proteolytic stability of synthetic peptides remains to be explored. In this study, a platform to enhance the proteolytic stability of peptides was developed by controllably dimerizing them into α-helical dimers, connected by two disulfide bonds. This platform not only directs peptides toward an α-helical conformation but permits control of the interfacial hydrophobic interactions between the peptides of the dimer. Using two model dimeric systems constructed from the N-terminal α-helix of RNase A and known inhibitors for the E3 ubiquitin ligase MDM2 (and its homologue MDMX), a deeper understanding into the interplay of disulfide bonds, α-helicity, and hydrophobic interactions on enhanced proteolytic stability was sought out. Results reveal that all three parameters play an important role on attaining ultrahigh proteolytic resistance, a concept that can be exploited for the development of future peptide therapeutics. The understanding gained through this study will enable this strategy to be tailored to new peptides because the proposed strategy displays substantial tolerance to sequence permutation. It thus appears promising for conveniently creating prodrugs composed entirely of the therapeutic peptide itself (i.e., in the form of a dimer).

The millisecond time scale motions in ribonuclease A (RNase A) were studied by solution NMR CPMG and off-resonance R1ρ relaxation dispersion experiments over a wide pH and temperature range. These experiments identify three separate protein regions termed Cluster 1, Cluster 2, and R33, whose motions are governed by distinct thermodynamic parameters. Moreover, each of these regions has motions with different pH dependencies. Cluster 1 shows an increase in activation enthalpy and activation entropy as the pH is lowered, whereas Cluster 2 exhibits the opposite behavior. In contrast, the activation enthalpy and entropy of R33 show no pH dependence. Compounding the differences, Δω values for Cluster 2 are characteristic of two-site conformational exchange, yet similar analysis for Cluster 1 indicates that this region of the enzyme exhibits conformational fluctuations between a major conformer and a pH-dependent average of protonated and deprotonated minor conformers.

The adsorption behavior of hard and soft proteins under the effect of an external electric field was investigated by a combination of spectroscopic ellipsometry and molecular dynamics (MD) simulations. Optically transparent carbon electrodes (OTCE) were used as conductive, sorbent substrates. Lysozyme (LSZ) and ribonuclease A (RNase A) were selected as representative hard proteins, whereas myoglobin (Mb), α-lactalbumin (α-LAC), bovine serum albumin (BSA), glucose oxidase (GOx), and immunoglobulin G (IgG) were selected to represent soft proteins. In line with recent publications from our group, the experimental results revealed that while the adsorption of all investigated proteins can be enhanced by the potential applied to the electrode, the effect is more pronounced for hard proteins. In contrast with the incomplete monolayers formed at open-circuit potential, the application of +800 mV to the sorbent surface induced the formation of multiple layers of protein. These results suggest that this effect can be related to the intrinsic polarizability of the protein (induction of dipoles), the resulting surface accessible solvent area (SASA), and structural rearrangements induced upon the incorporation on the protein layer. The described experiments are critical to understand the relationship between the structure of proteins and their tendency to form (under electric stimulation) layers with thicknesses that greatly surpass those obtained at open-circuit conditions.

Nanoparticles immersed in biological fluids readily adsorb proteins. The protein corona thus generated on the surface of the particles largely determines their biological fate. Since biological fluids, e.g., blood plasma, contain a large number of proteins, competitive adsorption must be considered. We study the competitive adsorption of lysozyme, cytochrome c, papain, and RNase A onto a soft charged polymeric layer. The experimental data of binary protein mixtures are compared to a theoretical model taking into account electrostatic and hydrophobic interactions between the proteins and the network. The interactions between bound proteins are modeled within a second virial approximation. The model possesses full generality and can be applied to the adsorption of an arbitrary number of protein types. The parameters describing the adsorption of a single protein type are obtained by isothermal titration calorimetry (ITC), while the competitive adsorption of a binary mixture is studied by fluorescence spectroscopy. The competitive adsorption can be predicted from the data related to the adsorption of the single types without adjustable parameters.

The dependence of static light scattering upon the compositions of solutions including hen egg white ovalbumin, hen egg white ovomucoid, ribonuclease A, and binary mixtures of these proteins at total concentrations of up to about 40 g/L were measured at different values of the pH and ionic strength. At the pH values of measurement, ovalbumin and ovomucoid have a net negative charge and ribonuclease A has a net positive charge. The observed dependence of scattering intensity upon solution composition may be accounted for by an extension of previously formulated equivalent hard particle models that allows for the presence of both repulsive interactions between like species and attractive interactions between unlike species in mixtures of positively and negatively charged proteins.

Ribonuclease A (RNase A) is a small globular enzyme that lyses RNA. The remarkable solution stability of its structure and enzymatic activity has led to its investigation to develop a new class of drugs for cancer chemotherapeutics. However, the successful clinical application of RNase A has been reported to be limited by insufficient stability and loss of enzymatic activity when it was coupled with a biomaterial carrier for drug delivery. The objective of this study was to characterize the structural stability and enzymatic activity of RNase A when it was adsorbed on different surface chemistries (represented by fused silica glass, high-density polyethylene, and poly(methyl-methacrylate)). Changes in protein structure were measured by circular dichroism, amino acid labeling with mass spectrometry, and in vitro assays of its enzymatic activity. Our results indicated that the process of adsorption caused RNase A to undergo a substantial degree of unfolding with significant differences in its adsorbed structure on each material surface. Adsorption caused RNase A to lose about 60% of its native-state enzymatic activity independent of the material on which it was adsorbed. These results indicate that the native-state structure of RNase A is greatly altered when it is adsorbed on a wide range of surface chemistries, especially at the catalytic site. Therefore, drug delivery systems must focus on retaining the native structure of RNase A in order to maintain a high level of enzymatic activity for applications such as antitumor chemotherapy.

We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea-protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome c do not undergo urea-induced transitions. The former remains folded, while the latter is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and α-chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea-protein interactions in both the native and unfolded states. We interpreted the urea-dependent volumetric properties of the proteins in terms of the equilibrium constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of k, ΔV0, and ΔKT0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea-protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent-protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides.

In peritoneal dialysis (PD), glucose degradation products (GDPs), which are formed during heat sterilization of dialysis fluids, lead to structural and functional changes in the peritoneal membrane, which eventually result in the loss of its ultrafiltration capacity. To determine the molecular mechanisms behind these processes, the present study tested the influence of the six major α-dicarbonyl GDPs in PD fluids, namely, glyoxal, methylglyoxal, 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), and glucosone with respect to their potential to impair the enzymatic activity of RNase A as well as their effects on cell viability. For comprehensive risk assessment, the α-dicarbonyl GDPs were applied separately and in concentrations as present in conventional PD fluids. Thus, it was shown that after 5 days, glucosone impaired RNase A activity most distinctly (58% remaining activity, p < 0.001 compared to that of the control), followed by 3,4-DGE (62%, p < 0.001), 3-DGal (66%, p < 0.001), and 3-DG (76%, p < 0.01). Methylglyoxal and glyoxal caused weaker inactivation with significant effects only after 10 days of incubation (79%, 81%, p < 0.001). Profiling of the advanced glycation end products formed during the incubation of RNase A with methylglyoxal revealed predominant formation of the arginine modifications imidazolinone, CEA/dihydroxyimidazoline, and tetrahydropyrimidine at Arg10, Arg33, Arg39, and Arg85. Particularly, modification at Arg39 may severely affect the active site of the enzyme. Additionally, structure- and concentration-specific assessment of the cytotoxicity of the α-dicarbonyl GDPs was performed. Although present at very low concentration, the cytotoxic effect of PD fluids after 2 days of incubation was exclusively caused by 3,4-DGE (14% cell viability, p < 0.001). After 4 days of incubation, 3-DGal (13% cell viability, p < 0.001), 3-DG (24%, p < 0.001), and, to a lower extent, glyoxal and methylglyoxal (both 57%, p < 0.01) also reduced cell viability significantly. In conclusion, 3,4-DGE, 3-DGal, and glucosone appear to be the most relevant parameters for the biocompatibility of PD fluids.

The adducts formed between trans-(dimethylamino)(methylamino)dichloridoplatinum(II), [t-PtCl2(dma)(ma)], and two model proteins, i.e., hen egg white lysozyme and bovine pancreatic ribonuclease, were independently characterized by X-ray crystallography and electrospray ionization mass spectrometry. In these adducts, the Pt(II) center, upon chloride release, coordinates either to histidine or aspartic acid residues while both alkylamino ligands remain bound to the metal. Comparison with the cisplatin derivatives of the same proteins highlights for [t-PtCl2(dma)(ma)] a kind of biomolecular metalation remarkably different from that of cisplatin.

Here we demonstrate that protein enzymes captured in the solvent channels of three-dimensional DNA crystals are catalytically active. Using RNase A as a model enzyme system, we show that crystals infused with enzyme can cleave a dinucleotide substrate with similar kinetic restrictions as other immobilized enzyme systems. This new vehicle for immobilized enzymes, created entirely from biomolecules, opens possibilities for developing modular solid-state catalysts that could be both biocompatible and biodegradable.

The crystal structure of the main adduct formed in the reaction between cisplatin and bovine pancreatic ribonuclease is reported here. Notably, in both of the protein molecules present in the asymmetric unit, platinum(II) binding takes place exclusively at the level of Met29. In one of the two molecules, the Gln28 side chain completes the platinum coordination sphere, anchoring the cisplatin fragment to the protein in a bidentate fashion. These results contain interesting implications for understanding the biological chemistry of this important drug.

The heat-induced desorption and adsorption of the proteins lysozyme, ribonuclease A, bovine serum albumin, and fibronectin at protein layers was investigated in two different environments: pure buffer and protein solution. Using two different environments allows us to distinguish between thermodynamic and kinetic mechanisms in the adsorption process. We observed a desorption in buffer and an adsorption in protein solution, depending upon protein properties, such as size, stability, and charge. We conclude that the desorption in buffer is mainly influenced by the mobility of the proteins at the interface, while the adsorption in protein solution is driven by conformational changes and, thereby, a gain in entropy. These results are relevant for controlling biofilm formation at solid-liquid interfaces.

The mechanism of the backbone cleavage-transesterification step of the RNase A enzyme remains controversial even after 60 years of study. We report quantum mechanics/molecule mechanics (QM/MM) free energy calculations for two optimized reaction paths based on an analysis of all structural data and identified by a search for reaction coordinates using a reliable quantum chemistry method (B3LYP), equilibrated structural optimizations, and free energy estimations. Both paths are initiated by nucleophilic attack of the ribose O2' oxygen on the neighboring diester phosphate bond, and both reach the same product state (PS) (a O3'-O2' cyclic phosphate and a O5' hydroxyl terminated fragment). Path 1, resembles the widely accepted dianionic transition-state (TS) general acid (His119)/base (His12) classical mechanism. However, this path has a barrier (25 kcal/mol) higher than that of the rate-limiting hydrolysis step and a very loose TS. In Path 2, the proton initially coordinating the O2' migrates to the nonbridging O1P in the initial reaction path rather than directly to the general base resulting in a triester (substrate as base) AN + DN mechanism with a monoanionic weakly stable intermediate. The structures in the transition region are associative with low barriers (TS1 10, TS2 7.5 kcal/mol). The Path 2 mechanism is consistent with the many results from enzyme and buffer catalyzed and uncatalyzed analog reactions and leads to a PS consistent with the reactive state for the following hydrolysis step. The differences between the consistently estimated barriers in Path 1 and 2 lead to a 10(11) difference in rate strongly supporting the less accepted triester mechanism.