Identification of the Acidification Mechanism of the Optimal pH for RNase He1.

Ribonuclease (RNase) He1 is a small ribonuclease belonging to the RNase T1 family. Most of the RNase T1 family members are active at neutral pH, except for RNase Ms, U2, and He1, which function at an acidic pH. We crystallized and analyzed the structure of RNase He1 and elucidated how the acidic amino residues of the α1ß3- (He1:26-33) and ß67-loops (He1:87-95) affect their optimal pH. In He1, Ms, and U2, the hydrogen bonding network formed by the acidic amino acids in the ß67-loop suggested that the differences in the acidification mechanism of the optimum pH specified the function of these RNases. We found that the amino acid sequence of the ß67-loop was not conserved and contributed to acidification of the optimum pH in different ways. Mutations in the acidic residues in He1 promoted anti-tumor growth activity, which clarified the role of these acidic amino residues in the binding pocket. These findings will enable the identification of additional targets for modifying pH-mediated enzymatic activities.

RNase T1 Refolding Assay for Determining Mitochondrial Cyclophilin D Activity: A Novel In Vitro Method Applicable in Drug Research and Discovery.

Human cyclophilin D is a mitochondrial peptidyl-prolyl isomerase that plays a role in regulating the opening of the mitochondrial permeability transition pore. It is considered a viable and promising molecular target for the treatment of diseases for which disease development is associated with pore opening, e.g., Alzheimer's disease or ischemia/reperfusion injury. Currently available and widely used in vitro methods based on Kofron's assay for determining cyclophilin D activity suffer from serious drawbacks and limitations. In this study, a completely novel approach for an in vitro assay of cyclophilin D activity using RNase T1 refolding is introduced. The method is simple and is more in line with the presumed physiological role of cyclophilin D in protein folding than Kofron's assay, which relies on a peptide substrate. The method is applicable for identifying novel inhibitors of cyclophilin D as potential drugs for the treatment of the diseases mentioned above. Moreover, the description of CypD activity in the in vitro RNase T1 refolding assay reveals new possibilities for investigating the role of cyclophilin D in protein folding in cells and may lead to a better understanding of its pathological and physiological roles.

Reactivity and Specificity of RNase T1, RNase A, and RNase H toward Oligonucleotides of RNA Containing 8-Oxo-7,8-dihydroguanosine.

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 T1, 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 T1 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.

Molecular Interpretation of Preferential Interactions in Protein Solvation: A Solvent-Shell Perspective by Means of Minimum-Distance Distribution Functions.

Preferential solvation is a fundamental parameter for the interpretation of solubility and solute structural stability. The molecular basis for solute-solvent interactions can be obtained through distribution functions, and the thermodynamic connection to experimental data depends on the computation of distribution integrals, specifically Kirkwood-Buff integrals for the determination of preferential interactions. Standard radial distribution functions, however, are not convenient for the study of the solvation of complex, nonspherical solutes, as proteins. Here we show that minimum-distance distribution functions can be used to compute KB integrals while at the same time providing an insightful view of solute-solvent interactions at the molecular level. We compute preferential solvation parameters for Ribonuclease T1 in aqueous solutions of urea and trimethylamine N-oxide (TMAO) and show that, while macroscopic solvation shows that urea is preferentially bound to the protein surface and TMAO is preferentially excluded, both display specific density augmentations at the protein surface in dilute solutions. Therefore, direct protein-osmolyte interactions can play a role in the stability and activity of the protein even for preferentially hydrated systems. The generality of the distribution function and its natural connection to thermodynamic data suggest that it will be useful in general for the study of solvation in mixtures of structurally complex solutes and solvents.

Stable Isotope Labeling for Improved Comparative Analysis of RNA Digests by Mass Spectrometry.

Even with the advent of high throughput methods to detect modified ribonucleic acids (RNAs), mass spectrometry remains a reliable method to detect, characterize, and place post-transcriptional modifications within an RNA sequence. Here we have developed a stable isotope labeling comparative analysis of RNA digests (SIL-CARD) approach, which improves upon the original 18O/16O labeling CARD method. Like the original, SIL-CARD allows sequence or modification information from a previously uncharacterized in vivo RNA sample to be obtained by direct comparison with a reference RNA, the sequence of which is known. This reference is in vitro transcribed using a 13C/15N isotopically enriched nucleoside triphosphate (NTP). The two RNAs are digested with an endonuclease, the specificity of which matches the labeled NTP used for transcription. As proof of concept, several transfer RNAs (tRNAs) were characterized by SIL-CARD, where labeled guanosine triphosphate was used for the reference in vitro transcription. RNase T1 digestion products from the in vitro transcript will be 15 Da higher in mass than the same digestion products from the in vivo tRNA that are unmodified, leading to a doublet in the mass spectrum. Singlets, rather than doublets, arise if a sequence variation or a post-transcriptional modification is present that results in a relative mass shift different from 15 Da. Moreover, the use of the in vitro synthesized tRNA transcript allows for quantitative measurement of RNA abundance. Overall, SIL-CARD simplifies data analysis and enhances quantitative RNA modification mapping by mass spectrometry. Graphical Abstract ᅟ.

Disulfide-bridging PEGylation during refolding for the more efficient production of modified proteins.

Proteins that are modified by chemical conjugation require at least two separate purification processes. First the bulk protein is purified, and then after chemical conjugation, a second purification process is required to obtain the modified protein. In an effort to develop new enabling technologies to integrate bioprocessing and protein modification, we describe the use of disulfide-bridging conjugation to conduct PEGylation during protein refolding. Preliminary experiments using a PEG-mono-sulfone reagent with partially unfolded leptin and unfolded RNAse T1 indicated that the cysteine thiols underwent disulfide-bridging conjugation to give the PEGylated proteins. Interferon-ß1b (IFN-ß1b) was then expressed in E.coli as inclusion bodies and found to undergo disulfide bridging-conjugation during refolding. The PEG-IFN-ß1b was isolated by ion-exchange chromatography and displayed in vitro biological activity. In the absence of the PEGylation reagent, IFN-ß1b refolding was less efficient and yielded protein aggregates. No PEGylation was observed if the cysteines on IFN-ß1b were first modified with iodoacetamide prior to refolding. Our results demonstrate that the simultaneous refolding and disulfide bridging PEGylation of proteins could be a useful strategy in the development of affordable modified protein therapeutics.

Probing water environment of Trp59 in ribonuclease T1: insight of the structure-water network relationship.

In this study, we used the tryptophan analogue, (2,7-aza)Trp, which exhibits water catalyzed proton transfer isomerization among N(1)-H, N(7)-H, and N(2)-H isomers, to probe the water environment of tryptophan-59 (Trp59) near the connecting loop region of ribonuclease Tl (RNase T1) by replacing the tryptophan with (2,7-aza)Trp. The resulting (2,7-aza)Trp59 triple emission bands and their associated relaxation dynamics, together with relevant data of 7-azatryptophan and molecular dynamics (MD) simulation, lead us to propose two Trp59 containing conformers in RNase T1, namely, the loop-close and loop-open forms. Water is rich in the loop-open form around the proximity of (2,7-aza)Trp59, which catalyzes (2,7-aza)Trp59 proton transfer in the excited state, giving both N(1)-H and N(7)-H isomer emissions. The existence of N(2)-H isomer in the loop-open form, supported by the MD simulation, is mainly due to the specific hydrogen bonding between N(2)-H proton and water molecule that bridges N(2)-H and the amide oxygen of Pro60, forming a strong network. The loop-close form is relatively tight in space, which squeezes water molecules out of the interface of α-helix and ß2 strand, joined by the connecting loop region; accordingly, the water-scant environment leads to the sole existence of the N(1)-H isomer emission. MD simulation also points out that the Trp-water pairs appear to preferentially participate in a hydrogen bond network incorporating polar amino acid moieties on the protein surface and bulk waters, providing the structural dynamic features of the connecting loop region in RNase T1.

Binase-like guanyl-preferring ribonucleases are new members of Bacillus PhoP regulon.

Extracellular low-molecular weight guanyl-preferring ribonucleases (LMW RNases) of Bacillus sp. comprise a group of hydrolytic enzymes that share highly similar structural and catalytic characteristics with barnase, a ribonuclease from Bacillus amyloliquefaciens, and binase, a ribonuclease from Bacillus intermedius. Although the physical-chemical and catalytic properties of Bacillus guanyl-preferring ribonucleases are very similar, there is considerably more variation in the environmental conditions that lead to the induction of the genes encoding these RNases. Based on structural differences of their genes the guanyl-preferring ribonucleases have been sub-divided into binase-like and barnase-like groups. Here we show the ability of the key regulator of phosphate deficiency response, PhoP, to direct the transcription of the binase-like RNases but not barnase-like RNases. These results, together with our demonstration that binase-like RNases are induced in response to phosphate starvation, allow us to categorise this group of ribonucleases as new members of Bacillus PhoP regulon. In contrast, the barnase-like ribonucleases are relatively insensitive to the phosphate concentration and the environmental conditions that are responsible for their induction, and the regulatory elements involved, are currently unknown.

X-ray crystallographic structure of RNase Po1 that exhibits anti-tumor activity.

RNase Po1 is a guanylic acid-specific ribonuclease member of the RNase T1 family from Pleurotus ostreatus. We previously reported that RNase Po1 inhibits the proliferation of human tumor cells, yet RNase T1 and other T1 family RNases are non-toxic. We determined the three-dimensional X-ray structure of RNase Po1 and compared it with that of RNase T1. The catalytic sites are conserved. However, there are three disulfide bonds, one more than in RNase T1. One of the additional disulfide bond is in the catalytic and binding site of RNase Po1, and makes RNase Po1 more stable than RNase T1. A comparison of the electrostatic potential of the molecular surfaces of these two proteins shows that RNase T1 is anionic whereas RNase Po1 is cationic, so RNase Po1 might bind to the plasma membrane electrostatically. We suggest that the structural stability and cationic character of RNase Po1 are critical to the anti-cancer properties of the protein.

Contribution of hydrogen bonds to protein stability.

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

The inhibition of human tumor cell proliferation by RNase Pol, a member of the RNase T1 family, from Pleurotus ostreatus.

RNase Po1 is a guanylic acid-specific ribonuclease (a RNase T1 family RNase) from Pleurotus ostreatus. We determined the cDNA sequence encoding RNase Po1 and expressed RNase Po1 in Escherichia coli. A comparison of the enzymatic properties of RNase Po1 and RNase T1 indicated that the optimum temperature for RNase Po1 activity was 20 °C higher than that for RNase T1. An MTT assay indicated that RNase Po1 inhibits the proliferation of human neuroblastoma cells (IMR-32 and SK-N-SH) and human leukemia cells (Jurkat and HL-60). Furthermore, Hoechst 33342 staining showed morphological changes in HL-60 cells due to RNase Po1, and flow cytometry indicated the appearance of a sub-G1 cell population. The extent of these changes was dependent on the concentration of RNase Pol. We suggest that RNase Po1 induces apoptosis in tumor cells.

Structure and function studies on enzymes with a catalytic carboxyl group(s): from ribonuclease T1 to carboxyl peptidases.

A group of enzymes, mostly hydrolases or certain transferases, utilize one or a few side-chain carboxyl groups of Asp and/or Glu as part of the catalytic machinery at their active sites. This review follows mainly the trail of studies performed by the author and his colleagues on the structure and function of such enzymes, starting from ribonuclease T1, then extending to three major types of carboxyl peptidases including aspartic peptidases, glutamic peptidases and serine-carboxyl peptidases.

The prolyl isomerase SlyD is a highly efficient enzyme but decelerates the conformational folding of a client protein.

Folding enzymes often use distinct domains for the interaction with a folding protein chain and for the catalysis of intrinsically slow reactions such as prolyl cis/trans isomerization. Here, we investigated the refolding reaction of ribonuclease T1 in the presence of the prolyl isomerase SlyD from Escherichia coli to examine how this enzyme catalyzes the folding of molecules with an incorrect trans proline isomer and how it modulates the conformational folding of the molecules with the correct cis proline. The kinetic analysis suggests that prolyl cis → trans isomerization in the SlyD-bound state shows a rate near 100 s(-1) and is thus more than 10(4)-fold accelerated, relative to the uncatalyzed reaction. As a consequence of its fast binding and efficient catalysis, SlyD retards the conformational folding of the protein molecules with the correct cis isomer, because it promotes the formation of the species with the incorrect trans isomer. In the presence of ≥1 µM SlyD, protein molecules with cis and trans prolyl isomers refold with identical rates, because SlyD-catalyzed cis/trans equilibration is faster than conformational folding. The cis or trans state of a particular proline is thus no longer a determinant for the rate of folding.

Small-angle X-ray scattering constraints and local geometry like secondary structures can construct a coarse-grained protein model at amino acid residue resolution.

We have developed a new methodology that determines protein structures using small-angle X-ray scattering (SAXS) data. The current bottlenecks in determining the protein structures require a new strategy using the simple design of an experiment, and SAXS is suitable for this purpose in spite of its low information content. First we demonstrated that SAXS constraints work additively to NMR-derived information in calculating structures. Next, structure calculations for nine proteins taking different folds were performed using the SAXS constraints combined with the NMR-derived distance restraints for local geometry such as secondary structures or those for tertiary structure. The results show that the SAXS constraints complemented the tertiary-structural information for all the proteins, and that accuracy of the structures thus obtained with SAXS constraints and local geometrical restraints ranged from 1.85 to 4.33Å. Based on these results, we were able to construct a coarse-grained protein model at amino acid residue resolution.

Reaction of singlet oxygen with tryptophan in proteins: a pronounced effect of the local environment on the reaction rate.

Singlet molecular oxygen, O(2)(a(1)Δ(g)), can influence many processes pertinent to the function of biological systems, including events that result in cell death. Many of these processes involve a reaction between singlet oxygen and a given amino acid in a protein. As a result, the behavior of that protein can change, either because of a structural alteration and/or a direct modification of an active site. Surprisingly, however, little is known about rate constants for reactions between singlet oxygen and amino acids when the latter are in a protein. In this report, we demonstrate using five separate proteins, each containing only a single tryptophan residue, that the rate constant for singlet oxygen reaction with tryptophan depends significantly on the position of this amino acid in the protein. Most importantly, the reaction rate constant depends not only on the accessibility of the tryptophan residue to oxygen, but also on factors that characterize the local molecular environment of the tryptophan in the protein. The fact that the local protein environment can either appreciably inhibit or accelerate the reaction of singlet oxygen with a given amino acid can have significant ramifications for singlet-oxygen-mediated events that perturb cell function.

Studying salt effects on protein stability using ribonuclease t1 as a model system.

Salt ions affect protein stability in a variety of ways. In general, these effects have either been interpreted from a charge solvation/charge screening standpoint or they have been considered to be the result of ion-specific interactions with a particular protein. Recent theoretical work suggests that a major contribution to salt effects on proteins is through the interaction of salt ions that are located near the protein surface and their induced point image charges that are located in the low-dielectric protein cavity. These interactions form the basis of "salting-out" interactions. Salt ions induce an image charge of the same sign in the low dielectric protein medium. The interaction between the induced charge and its mirror charge is repulsive and consequently thermodynamically destabilizing. However, a folded protein that has a much smaller surface area will be less destabilized than the unfolded state. Consequently, the folded state will be stabilized relative to the unfolded state. This work analyzes salt effects in the model enzyme ribonuclease t1, and demonstrates that interactions between salt ions and their induced point charges provide a major contribution to the observed salt-induced increase in protein stability. This work also demonstrates that in the case of weakly-binding ions (ions with binding constants that are in the order of 50 M(-1) and less), salting-out effects should still be considered in order to provide a more realistic interpretation of ion binding. These results should therefore be considered when salt effects are used to analyze electrostatic contributions to protein structure or are used to study the thermodynamics of proteins associated with halophillic organisms.

Transient enzyme-substrate recognition monitored by real-time NMR.

Slow protein folding processes during which kinetic folding intermediates occur for an extended time can lead to aggregation and dysfunction in living cells. Therefore, protein folding helpers have evolved, which prevent proteins from aggregation and/or speed up folding processes. In this study, we present the structural characterization of a long-living transient folding intermediate of RNase T1 (S54G/P55N) by time-resolved NMR spectroscopy. NMR resonances of this kinetic folding intermediate could be assigned mainly by a real-time 3D BEST-HNCA. These assignments were the basis to investigate the interaction sites between the protein folding helper enzyme SlyD(1-165) (SlyD*) from Escherichia coli (E. coli) and this kinetic intermediate at a residue resolution. Thus, we investigated the Michaelis-Menten complex of this enzyme reaction, because the NMR data acquisition was performed during the actual catalysis. The interaction surface of the transient folding intermediate is restricted to a region around the peptidyl-prolyl bond (Y38-P39), whose isomerization is catalyzed by SlyD*. The interaction surface regarding SlyD* extends from specific amino acids of the FKBP domain forming the peptidyl-prolyl cis/trans-isomerase active site to almost the entire IF domain. This illustrates an effective interplay between the two functional domains of SlyD* to facilitate protein folding catalysis.

Contribution of hydrophobic interactions to protein stability.

Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6±0.3 kcal/mol per -CH(2)- group), than to the stability of a large protein, VlsE (1.6±0.3 kcal/mol per -CH(2)- group). (2) Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kilocalories per mole) Phe18 (3.9), Met13 (3.1), Phe7 (2.9), Phe11 (2.7), and Leu21 (2.7). (3) Based on the Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a -CH(2)- group on folding contributes, on average, 1.1±0.5 kcal/mol to protein stability. (4) The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔG(tr) values from water to cyclohexane. (5) For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60±4% and hydrogen bonds contribute 40±4% to protein stability. (6) Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominantly by hydrophobic interactions.

Electrically-assisted delivery of an anionic protein across intact skin: cathodal iontophoresis of biologically active ribonuclease T1.

Cathodal iontophoresis of anionic macromolecules has been considered a major challenge owing to (i) the presence of a negative charge on the skin under physiological conditions and (ii) the electroosmotic solvent flow in the (opposite) anode-to-cathode direction. Moreover, electroosmosis, and not electromigration, was considered as the likely electrotransport mechanism for high molecular weight cations. However, it was recently shown that electromigration governed anodal iontophoretic transport of Cytochrome c (12.4 kDa) and Ribonuclease A (RNAse A; 13.6 kDa). Thus, the objective of this study was to investigate the feasibility of iontophoresing a negatively charged protein, the enzyme Ribonuclease T1 (RNAse T1, 11.1 kDa), from the cathode across intact skin. Cumulative permeation and skin deposition of RNAse T1 were investigated as a function of current density (0.15, 0.3 and 0.5 mA/cm(2) applied for 8h) using porcine ear skin and quantified by an enzymatic activity assay. Although RNAse T1 permeation was dependent upon current density (22.41 ± 8.10, 76.41 ± 56.98 and 142.19 ± 62.23µg/cm(2), respectively), no such relationship was observed with respect to skin deposition (9.78 ± 2.39, 7.76 ± 4.34 and 8.70 ± 2.94 µg/cm(2), respectively). MALDI-TOF spectra and the activity assay confirmed that RNAse T1 retained structural integrity and enzymatic function post-iontophoresis. Acetaminophen iontophoresis demonstrated the anode-to-cathode directionality of electroosmotic solvent flow confirming that RNAse T1 electrotransport was due entirely to electromigration. Interestingly, despite its lower net charge and higher molecular weight, electromigration of cationic Ribonuclease A was superior to that of RNAse T1 after iontophoresis at 0.5 mA/cm(2) for 8h. These results provide further evidence that charge to mass ratio and hence electric mobility might not alone be sufficient to predict protein electrotransport across the skin; three dimensional structures and the spatial distribution of physicochemical properties must also be considered. The skin extraction data suggest that negatively charged molecules may have fewer potential binding sites in the skin than their cationic counterparts. This was supported by confocal laser scanning microscopy images which showed that whereas fluorescence from RNAse A was distributed throughout the epidermis and dermis, RNAse T1 appeared to be bound to the epidermis alone. In conclusion, this is the first report demonstrating successful non-invasive cathodal iontophoresis of a negatively charged functional protein (RNAse T1) across intact skin.

Probing the effect of water-water interactions on enzyme activity with salt gradients: a case-study using ribonuclease t1.

Water molecules interact with one another via hydrogen bonds. Experimental and theoretical evidence indicates that these hydrogen bonds occur in two modalities--high- and low-angle hydrogen bonding--and that the addition of various solutes to water affects only the number of water molecules participating in a specific type of hydrogen bond interactions, not the nature of the water-water interactions. In this work, we have investigated the effect of each of these hydrogen bonding types upon the activity of the enzyme ribonuclease t1. This was done through perturbation of the water hydrogen bonding distribution by using various salts. Our results indicate that various salts differ in their ability to reduce the enzymatic activity of ribonuclease t1, and this ability is well correlated with the ability of each salt to promote high-angle hydrogen bonding in water. By applying the two-phase model of liquid water (i.e., liquid water being modeled as an equilibrium existing between two phases, LD and HD water), we demonstrate that our results are compatible with the assumption that increasing the population of high-angle hydrogen bonds among water molecules stabilizes the more compact, less active conformations of the enzyme. This indicates that the structures that proteins adopt in water solution depend upon the nature of interactions between water molecules.