Ribonuclease A Research Articles

Ligand-Induced Variations in Structural and Dynamical Properties Within an Enzyme Superfamily.

Enzyme catalysis is a complex process involving several steps along the reaction coordinates, including substrate recognition and binding, chemical transformation, and product release. Evidence continues to emerge linking the functional and evolutionary role of conformational exchange processes in optimal catalytic activity. Ligand binding changes the conformational landscape of enzymes, inducing long-range conformational rearrangements. Using functionally distinct members of the pancreatic ribonuclease superfamily as a model system, we characterized the structural and conformational changes associated with the binding of two mononucleotide ligands. By combining NMR chemical shift titration experiments with the chemical shift projection analysis (CHESPA) and relaxation dispersion experiments, we show that biologically distinct members of the RNase superfamily display discrete chemical shift perturbations upon ligand binding that are not conserved even in structurally related members. Amino acid networks exhibiting coordinated chemical shift displacements upon binding of the two ligands are unique to each of the RNases analyzed. Our results reveal the contribution of conformational rearrangements to the observed chemical shift perturbations. These observations provide important insights into the contribution of the different ligand binding specificities and effects of conformational exchange on the observed perturbations associated with ligand binding for functionally diverse members of the pancreatic RNase superfamily.

NaPi/S<sub>X</sub>-RNase segregates as a functional S-RNase and is induced under phosphate deficiency in Nicotiana alata

In plants, class III T2 RNases involves two groups of structurally similar proteins, but with different biological functions: S-RNases and non-S-RNases. S-RNases have been involved in self-incompatibility whereas non-S-RNases have been implicated in stress responses. Here we report a novel class III RNase termed NaPi/Sx-RNase, which works both in self-incompatibility and in response to phosphate deficiency. The NaPi/Sx-RNase gene was identified in roots of Nicotiana alata grown in the absence of inorganic phosphate. Phylogenetic analysis showed that NaPi/Sx-RNase was included within the class III RNase T2 group. The NaPi/Sx-RNase was expressed in styles and its temporal expression increased in parallel to stylar development, with a slight decrease after anthesis. Progeny analysis showed that NaPi/Sx-RNase and S107-RNase, a functional allele of the self-incompatibility system, segregated in a 1:1 ratio. The progeny segregation of a semicompatible cross, in which NaPi/Sx-RNase was shared by the two parents, exhibited a pattern consistent with a functional S-RNase allele. Considering genetic segregation, primary structure, and physiological role, the NaPi/Sx-RNase may be either an S-RNase with diversified functions or a non-S-RNase linked to the S-locus. To our knowledge, this is the first evidence for a specific function of the S-locus other than the self-incompatibility reaction. These results support the hypothesis that the self-incompatibility and stress responses may have evolved from a common origin.

A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance.

The past decade has seen a tremendous increase in interest and progress in the field of sperm epigenetics. Studies have shown that chromatin regulation during male germline development is multiple and complex, and that the spermatozoon possesses a unique epigenome. Its DNA methylation profile, DNA-associated proteins, nucleo-protamine distribution pattern and non-coding RNA set up a unique epigenetic landscape which is delivered, along with its haploid genome, to the oocyte upon fertilization, and therefore can contribute to embryogenesis and to the offspring health. An emerging body of compelling data demonstrates that environmental exposures and paternal lifestyle can change the sperm epigenome and, consequently, may affect both the embryonic developmental program and the health of future generations. This short review will attempt to provide an overview of what is currently known about sperm epigenome and the existence of transgenerational epigenetic inheritance of paternally acquired traits that may contribute to the offspring phenotype.

New insights into hypoxia-related mechanisms involved in different microvascular patterns of bronchopulmonary carcinoids and poorly differentiated neuroendocrine carcinomas. Role of ribonuclease T2 (RNASET2) and HIF-1α

Ribonuclease T2 (RNASET2) is a pleiotropic and polyfunctional protein, which exerts several different activities in neoplastic cells since the early steps of tumor development. Besides having an antitumorigenic activity, RNASET2 inhibits both bFGF-induced and VEGF-induced angiogenesis and has a role as a stress-response, alarmin-like, protein. In this study, we investigated RNASET2 expression in well-differentiated and poorly differentiated neuroendocrine neoplasms of the lung (Lu-NENs), which are known to show clear-cut differences in morphology, biology and clinical behavior. In addition, we explored possible relationships between RNASET2 expression and a series of immunohistochemical markers related to hypoxic stress, apoptosis, proliferation and angiogenesis. Our results showed a significantly higher expression of RNASET2, HIF-1α, and its target CA IX in poorly differentiated than in well-differentiated Lu-NENs, the former also showing higher proliferation and apoptotic rates, as well as a lower microvessel density (MVD) than the latter. Moreover, we were able to demonstrate in vitro an overexpression of RNASET2 in consequence of the activation of HIF-1α. In conclusion, we suggest that in poorly differentiated Lu-NENs, RNASET2 expression may be induced by HIF-1α, behaving as an alarmin-like molecule. In this aggressive group of cancers, which have highly deregulated proliferation pathways, RNASET2 fails to exert the growth-inhibiting effects described in other types of neoplasms. Its increased expression, however, may contribute to the typical phenotypic alterations seen in poorly differentiated Lu-NENs, such as the high apoptotic rate and the extensive necrosis, and may also enhance the low MVD observed in these neoplasms.

Preliminary investigation of deoxyoligonucleotide binding to ribonuclease A using mass spectrometry: An attempt to develop a lab experience for undergraduates.

Deoxyoligonucleotide binding to bovine pancreatic ribonuclease A (RNase A) was investigated using electrospray ionization ion-trap mass spectrometry (ESI-IT-MS). Deoxyoligonucleotides included CCCCC (dC 5) and CCACC (dC 2AC 2). This work was an attempt to develop a biochemistry lab experience that would introduce undergraduates to the use of mass spectrometry for the analysis of protein-ligand interactions. Titration experiments were performed using a fixed RNase A concentration and variable deoxyoligonucleotide concentrations. Samples at equilibrium were infused directly into the mass spectrometer under native conditions. For each deoxyoligonucleotide, mass spectra showed one-to-one binding stoichiometry, with marked increases in the total ion abundance of ligand-bound RNase A complexes as a function of concentration, but the accurate determination of dC 5 and dC 2AC 2 dissociation constants was problematic.

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.

Preliminary investigation of deoxyoligonucleotide binding to ribonuclease A using mass spectrometry: An attempt to develop a lab experience for undergraduates

Deoxyoligonucleotide binding to bovine pancreatic ribonuclease A (RNase A) was investigated using electrospray ionization ion-trap mass spectrometry (ESI-IT-MS). Deoxyoligonucleotides included CCCCC (dC 5) and CCACC (dC 2AC 2). This work was an attempt to develop a biochemistry lab experience that would introduce undergraduates to the use of mass spectrometry for the analysis of protein-ligand interactions. Titration experiments were performed using a fixed RNase A concentration and variable deoxyoligonucleotide concentrations. Samples at equilibrium were infused directly into the mass spectrometer under native conditions. For each deoxyoligonucleotide, mass spectra showed one-to-one binding stoichiometry, with marked increases in the total ion abundance of ligand-bound RNase A complexes as a function of concentration, but the accurate determination of dC 5 and dC 2AC 2 dissociation constants was problematic.

RNASET2 impairs the sperm motility via PKA/PI3K/calcium signal pathways.

Asthenozoospermia is one of the leading causes of male infertility owing to a decline in sperm motility. Herein, we determined if there is a correlation between RNASET2 content on human spermatozoa and sperm motility in 205 semen samples from both asthenozoospermia patients and normozoospermia individuals. RNASET2 content was higher in sperm from asthenozoospermia patients than in normozoospermia individuals. On the other hand, its content was inversely correlated with sperm motility as well as progressive motility. Moreover, the inhibitory effect of RNASET2 on sperm motility was induced by incubating normozoospermic sperm with RNase T2 protein. Such treatment caused significant declines in intracellular spermatozoa PKA activity, PI3K activity and calcium level, which resulted in severely impaired sperm motility, and the sperm motility was largely rescued by cAMP supplementation. Finally, protein immunoprecipitation and mass spectrometry identified proteins whose interactions with RNASET2 were associated with declines in human spermatozoa motility. AKAP4, a protein regulating PKA activity, coimmunoprecipated with RNASET2 and they colocalized with one another in the sperm tail, which might contribute to reduced sperm motility. Thus, RNASET2 may be a novel biomarker of asthenozoospermia. Increases in RNASET2 can interact with AKAP4 in human sperm tail and subsequently reduce sperm motility by suppressing PKA/PI3K/calcium signaling pathways.

Improved RNA‐modification mapping through employment of novel ribonucleases and LC‐MS

More than 150 different chemical modifications have been documented in various types of ribonucleic acids (RNA) [1]. They play key roles in translation, cellular fate, and stabilization of RNA structure [2]. While majority of these modifications are catalyzed by cellular enzymes, others are generated by xenobiotic stress exposure. RNA modifications have been linked to human diseases [3] such as neurological disorders and cancer.Understanding the role of these modifications in human disorders requires precise identification and location of the site in the overall RNA sequence. Nucleobase specific ribonuclease (RNase) enzymes are useful in mapping the location of chemical modification. Here the RNA is digested with a nucleobase‐specific enzyme and the resulting digestion products are subjected to liquid chromatography coupled with mass spectrometry (LC‐MS) ‐based sequencing, a method widely referred to as RNA modification mapping [4].Commercially available RNase T1 (G‐specific) and RNase A (pyrimidine‐specific) have been widely used in RNA modification mapping procedures. To augment the limitations of these enzymes, additional nucleobase‐specific enzymes, RNase MC1 (U‐specific) [5] and cusativin (C‐specific) [6] have been developed that could complement the existing enzymes. Thus, the digestion products generated by multiple enzymes can complement each other thereby improving the sequence coverage and generation of overlapping digestion products of substrate RNA. In the current investigation, we will present reconstruction of modified tRNA sequences from the digestion products generated by digestion of Escherichia coli tRNA by multiple nucleobase‐specific enzymes. We will also demonstrate the utility of this technology to map the stress‐induced damage in the ribosomal RNA. The advantage of such a multi‐pronged approach as opposed to the single enzyme usage in reconstruction of modified RNA sequences will be discussed.Support or Funding InformationFinancial support for this work was provided by the National Institutes of Health (GM058843) and the University of Cincinnati.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiogenin/Ribonuclease 5 Is an EGFR Ligand and a Serum Biomarker for Erlotinib Sensitivity in Pancreatic Cancer

Pancreatic ribonuclease (RNase) is a secreted enzyme critical for host defense. We discover an intrinsic RNase function, serving as a ligand for epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase (RTK), in pancreatic ductal adenocarcinoma (PDAC). The closely related bovine RNase A and human RNase 5 (angiogenin [ANG]) can trigger oncogenic transformation independently of their catalytic activities via direct association with EGFR. Notably, high plasma ANG level in PDAC patients is positively associated with response to EGFR inhibitor erlotinib treatment. These results identify a role of ANG as a serum biomarker that may be used to stratify patients for EGFR-targeted therapies, and offer insights into the ligand-receptor relationship between RNase and RTK families.

Preliminary investigation of deoxyoligonucleotide binding to ribonuclease A using mass spectrometry: An attempt to develop a lab experience for undergraduates.

Deoxyoligonucleotide binding to bovine pancreatic ribonuclease A (RNase A) was investigated using electrospray ionization ion-trap mass spectrometry (ESI-IT-MS). Deoxyoligonucleotides included CCCCC (dC 5) and CCACC (dC 2AC 2). This work was an attempt to develop a biochemistry lab experience that would introduce undergraduates to the use of mass spectrometry for the analysis of protein-ligand interactions. Titration experiments were performed using a fixed RNase A concentration and variable deoxyoligonucleotide concentrations. Samples at equilibrium were infused directly into the mass spectrometer under native conditions. For each deoxyoligonucleotide, mass spectra showed one-to-one binding stoichiometry, with marked increases in the total ion abundance of ligand-bound RNase A complexes as a function of concentration, but the accurate determination of dC 5 and dC 2AC 2 dissociation constants was problematic.

MicroRNA hsa-let-7e-5p as a potential prognosis marker for rectal carcinoma with liver metastases.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that target mRNAs for translational repression or cleavage. The present study was conducted to identify differentially expressed miRNAs in primary tumor tissues of rectal carcinoma (RC) that may be associated with heterochrony hepatic metastasis (HHM). Samples were collected exclusively from patients with RC but not colon cancer (CC); Next-generation high-throughput sequencing technology and bioinformatics tools were used to profile and analyze small RNAs and their corresponding targets in primary tumor tissues with HHM (n=2) or without metastases (non-metastatic, NM; n=2). A total of 24 known miRNAs were identified to be differentially expressed (P<0.01; absolute value of log2-fold change ≥1). Hsa-let-7e-5p exhibited the most significant elevation in tissues with HHM (log2-fold change=2.62). By combining online informatics resources and previous mRNA sequencing data, it was identified that 54 validated target genes of let-7e were downregulated in primary tumor tissues with HHM. A number of these target genes have been demonstrated to be directly involved in tumor metastasis (including MYC proto-oncogene, bHLH transcription factor, high-mobility group AT-Hook 2, peptidase inhibitor 3, KIT proto-oncogene receptor tyrosine kinase, Jun proto-oncogene, AP-1 transcription factor subunit and ribonuclease T2), or have physiological associations to immunity (including C-C motif chemokine receptor 4 and cluster of differentiation 40 ligand) and cellular metabolism (including peroxisome proliferator-activated receptor γ, coactivator 1 α). Next, 14 target genes were selected for reverse transcription-quantitative polymerase chain reaction analysis in non-sequenced samples, and the downregulation of 10 target genes in RC samples with HHM was confirmed. In addition, it was demonstrated that hsa-let-7e-5p stimulated colorectal cancer cell migration in vitro. The miRNA hsa-let-7e-5p may serve as a potential biomarker for rectal carcinoma-associated HHM, facilitating the identification of patients with RC who are at risk of developing HHM.

Five-Coordinate Platinum(II) Compounds Containing Sugar Ligands: Synthesis, Characterization, Cytotoxic Activity, and Interaction with Biological Macromolecules

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.

Updates in the Development of ImmunoRNases for the Selective Killing of Tumor Cells.

Targeted cancer therapy includes, amongst others, antibody-based delivery of toxic payloads to selectively eliminate tumor cells. This payload can be either a synthetic small molecule drug composing an antibody-drug conjugate (ADC) or a cytotoxic protein composing an immunotoxin (IT). Non-human cytotoxic proteins, while potent, have limited clinical efficacy due to their immunogenicity and potential off-target toxicity. Humanization of the cytotoxic payload is essential and requires harnessing of potent apoptosis-inducing human proteins with conditional activity, which rely on targeted delivery to contact their substrate. Ribonucleases are attractive candidates, due to their ability to induce apoptosis by abrogating protein biosynthesis via tRNA degradation. In fact, several RNases of the pancreatic RNase A superfamily have shown potential as anti-cancer agents. Coupling of a human RNase to a humanized antibody or antibody derivative putatively eliminates the immunogenicity of an IT (now known as a human cytolytic fusion protein, hCFP). However, RNases are tightly regulated in vivo by endogenous inhibitors, controlling the ribonucleolytic balance subject to the cell’s metabolic requirements. Endogenous inhibition limits the efficacy with which RNase-based hCFPs induce apoptosis. However, abrogating the natural interaction with the natural inhibitors by mutation has been shown to significantly enhance RNase activity, paving the way toward achieving cytolytic potency comparable to that of bacterial immunotoxins. Here, we review the immunoRNases that have undergone preclinical studies as anti-cancer therapeutic agents.

Raman-markers of X-ray radiation damage of proteins

Despite their high relevance, the mechanisms of X-ray radiation damage on protein structure yet have to be completely established. Here, we used Raman microspectrophotometry to follow X-ray-induced chemical modifications on the structure of the model protein bovine pancreatic ribonuclease (RNase A). The combination of dose-dependent Raman spectra and ultrahigh resolution (eight structures solved using data collected between 0.85 and 1.17 Å resolution on the same single crystal) allowed direct observation of several radiation damage events, including covalent bond breakages and formation of radicals. Our results are relevant for analytical photodamage detection and provide implications for a detailed understanding of the mechanisms of photoproduct formation.

Molecular mechanisms underlying the participation of Ribonuclease T2 gene into self-incompatibility of Citrus grandis var. Shatianyu Hort.

The RIB (ribonuclease T2) of Citrus grandis var. Shatianyu Hort involved in self-incompatibility (SI) mechanism was identified by prokaryotic expression. RT-qPCR results showed that the expression level of RIB in self pollinated stigma is significantly higher than that in cross pollinated stigma. A vector for prokaryotic expression of RIB was constructed after codon-optimization, and the recombinant protein was induced and purified. In vitro pollen germination test indicated that the RIB protein markedly inhibited pollen germination and pollen tube growth. The result is helpful for better understanding of the molecular mechanism underlying the SI in C. grandis.

Evaluation of Different Signal Peptides for Secretory Production of Recombinant Bovine Pancreatic Ribonuclease A in Gram Negative Bacterial System: An In silico Study

Background: Prokaryotic systems such as E. coli are among the most affordable and simplest hosts which are being employed to express recombinant proteins, nevertheless without appropriate signal peptide these systems cannot be used for secretory proteins. Bovine pancreatic ribonuclease A is a protein with four disulfide bonds which might be used as an immunoenzyme for immunotherapy. Consequently, the production of this recombinant protein, using prokaryotic system, requires a suitable signal peptide to protect disulfide bonds and to prevent misfolding. Objective: This study was designed to predict the best signal peptides to express bovine pancreatic ribonuclease A protein in E. coli. Method: In this study, 42 signal sequences were selected from data bases and the most important features of them were evaluated. First, n, h and c regions of signal peptides and their probability were investigated by signalP software’s version 4.1. Then, physico-chemical features of them were evaluated by Portparam and SOLpro. Also, secretion sorting and sub-cellular localization sites were evaluated by PRED-TAT and ProtcompB software programs. Results: The results showed that among all studied signal peptides only 28 out of 41 remained signal peptides could be considered as appropriate secretory signal peptides. Conclusion: Finally, Phage shock protein E, ranked as the best signal peptide and after that, pectate lyase B, F41 fimbrial protein and Lipopolysaccharide export system protein lptA considered as the next best signal peptides which were approved by in silico tools as the most appropriate secretory signal peptides in E. coli. However, further experiments are required to validate these in silico results. Keywords: Bacterial system, bioinformatics, bovine pancreatic ribonuclease A, E. coli, In silico, signal peptide.

Reactions of a tetranuclear Pt-thiosemicarbazone complex with model proteins

The tetranuclear Pt complex (PtL)4 (where L2− is the anion derived from para-isopropyl thiosemicarbazone) was first described in A.G. Quiroga et al., J. Med. Chem. 41, 1998, 1399–1408. (PtL)4 manifests antiproliferative properties toward various cancer cell lines being a promising anticancer drug candidate. Yet, details of its reactivity with biomolecules have not been elucidated. To this end, we investigated the reactions of (PtL)4 with a few model proteins, i.e. bovine pancreatic ribonuclease (RNase A), cytochrome c (Cyt c) and hen egg white lysozyme (Lysozyme), through electrospray ionization mass spectrometry and other biophysical methods. A rich reactivity of (PtL)4 with the above-mentioned model proteins is observed, leading to the formation of numerous metallodrug-protein adducts. The tetranuclear complex breaks down and various fragments bind proteins up to high metal/protein ratios; this typically results into very complicated mass spectral patterns. However, some of the main mass peaks could be assigned in the case of the Lysozyme adduct. In addition, crystallographic data were obtained for the (PtL)4/Lysozyme and (PtL)4/RNase A adducts pointing at His side chains as the primary binding sites for monometallic Pt fragments. Notably, a few selected features of the interactions observed in the (PtL)4/protein adducts were reproduced by reacting (PtL)4 with a small molecule, i.e. N-methylimidazole. In conclusion, the present study confirms the prodrug nature of the tetraplatinum complex, clarifies one possible pathway for its activation through cluster disassembly and allows initial identification of adducts formed with a representative protein.

Poly A tail length analysis of in vitro transcribed mRNA by LC-MS.

The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3' end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3' end can be locked in place by having a G or C after the poly nucleotide region. Graphical abstract Determination of mRNA poly A tail length using magnetic beads and LC-MS.

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.