Poly(propylene imine) dendrimers can bind to PEGylated albumin at PEG and albumin surface: Biophysical examination of a PEGylated platform to transport cationic dendritic nanoparticles

Cationic dendrimers are considered one of the best drug transporters in the body. However, in order to improve their biocompatibility, modification of them is required to reduce toxicity. In this way, many dendrimers may lose their original properties, for example, anticancer. To improve biocompatibility of dendrimers, it is possible to complex them with albumin, as is done very often in drug delivery. However, the interaction of dendrimers with albumin can lead to protein structure disruption or no complexation at all. Therefore, the investigation of the interaction between cationic poly-(propylene imine) dendrimers and polyethylene glycol (PEG)-albumin by fluorescence, circular dichroism, small angle X-ray scattering (SAXS), and transmission electron microscopy was carried out. Results show that cationic dendrimers bind to PEGylated albumin at PEG and albumin surfaces. The obtained results for 5k-PEG indicate a preferential binding of the dendrimers to PEG. For 20k-PEG binding of dendrimers to PEG and protein could induce a collapse of the PEG chain onto the protein surface. This opens up new possibilities to the use of PEGylated albumin as a platform to carry dendrimers without changing the albumin structure and improve the pharmacokinetic properties of dendrimers without further modification.     

NMR as a tool for elucidating the structures of circular and knotted proteins

Cyclotides are a recently discovered family of mini-proteins that have a head-to-tail cyclised backbone stabilized by a knotted arrangement of three disulfide bonds. They have a wide range of biological activities, including uterotonic, anti-bacterial, anti-HIV, and anti-tumour activity but their insecticidal activities suggest that their natural function is in plant defense. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. This stability and resistance to proteolysis makes them a potentially valuable protein engineering tool at the interface of chemistry and biology: they have the structure of proteins but the stability and biophysical properties of organic molecules. In this review the role of NMR in defining the structures of cyclotides is described.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: NMR studies of their tertiary and quaternary structures

Diruthenium-substituted Ru-Fe hybrid hemoglobins (Hb) were synthesized by heme substitution from protoheme to ruthenium (II) carbonyldeuteroporphyrin in the alpha or beta subunits. As the carbon monoxide coordinated to ruthenium (II) is not released under physiological conditions, deoxygenated Ru-Fe hybrid derivatives [alpha(Fe)2 beta(Ru-CO)2 and alpha(Ru-CO)2 beta(Fe)2] can serve as models for half-liganded Hbs. On the basis of proton NMR spectra of hyperfine-shifted proton resonances, these Ru-Fe hybrid Hbs have only small structural changes in the heme environment of the partner subunits at low pH. The proton NMR spectra of the intersubunit hydrogen-bonded protons also showed that the quaternary structures of the two complementary hybrids both remain in the "T-like state" at low pH, suggesting that the T to R structural conversion is induced by ligation of the third ligand molecule. Marked conformational changes in the heme vicinity are observed at high pH only for alpha(Ru-CO)2 beta(Fe)2, and its quaternary structure is converted into the "R state"; the alpha(Fe)2 beta(Ru-CO)2 hybrid does not undergo this change. This implies that the free-energy difference between the two quaternary states is smaller in the alpha-liganded hybrid than in the beta-liganded one.     

Rosin Nanoparticles as a Drug Delivery Carrier for the Controlled Release of Hydrocortisone

Hydrocortisone (HC)-loaded rosin nanoparticles were prepared by a dispersion and dialysis method without addition of surfactant. They were spherical: 167–332 nm diam. The drug was loaded approximately 50% of initial feeding amount in all formulation. Release of hydrocortisone from the nanoparticles in vitro gradually decreased with increasing initial rosin content at pH 7.4. HC was also released very slowly at pH 1.2. Nanoparticles based on rosin thus are potentially useful as a drug delivery system.     

Protein-protein interactions as a tool for site-specific labeling of proteins

Probing structures and dynamics within biomolecules using ensemble and single-molecule fluorescence resonance energy transfer requires the conjugation of fluorophores to proteins in a site-specific and thermodynamically nonperturbative fashion. Using single-molecule fluorescence-aided molecular sorting and the chymotrypsin inhibitor 2-subtilisin BPN' complex as an example, we demonstrate that protein-protein interactions can be exploited to afford site-specific labeling of a recombinant double-cysteine variant of CI2 without the need for extensive and time-consuming chromatography. The use of protein-protein interactions for site-specific labeling of proteins is compatible with and complementary to existing chemistries for selective labeling of N-terminal cysteines, and could be extended to label multiple positions within a given polypeptide chain.     

Attenuated total reflection IR spectroscopy as a tool to investigate the orientation and tertiary structure changes in fusion proteins

Membrane fusion proceeds via a merging of two lipid bilayers and a redistribution of aqueous contents and bilayer components. It involves transition states in which the phospholipids are not arranged in bilayers and in which the monolayers are highly curved. Such transition states are energetically unfavourable since biological membranes are submitted to strong repulsive hydration electrostatic and steric barriers. Viral membrane proteins can help to overcome these barriers. Viral proteins involved in membrane fusion are membrane associated and the presence of lipids restricts drastically the potential of methods (RMN, X-ray crystallography) that have been used successfully to determine the tertiary structure of soluble proteins. We describe here how IR spectroscopy allows to solve some of the problems related to the lipid environment.The principles of the method, the experimental setup and the preparation of the samples are briefly described. A few examples illustrate how attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy can be used to gain information on the orientation and the accessibility to the water phase of the fusogenic domain of viral proteins. Recent developments suggest that the method could also be used to detect changes located in the membrane domains and to identify intermediate structural states involved in the fusion process.     

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein–protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand–protein interaction sites, such as oligonucleotide–protein, drug–protein and protein–protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.     

Tritium planigraphy: A tool for studying the spatial structure of proteins and their complexes

The review is devoted to tritium planigraphy and its applications in solving a broad scope of problems in modern molecular and physicochemical biology. The method is based on nonselective substitution of tritium for hydrogen in the hydrocarbon parts of target molecules. It furnishes information on the steric accessibility of the components of a system under study (macromolecule within a complex amino acid residues, and even separate atomic groupings in a macromolecule) that characterizes the structure of the entire object. The technique is applicable to specimens in different phase states and has no limitations in respect of the target molecular mass. Tritium planigraphy is especially important in the cases when the biological macromolecules cannot be examined by the conventional methods (X-ray analysis and NMR spectroscopy). The review summarizes the studies of protein accessible surface and spatial arrangement, and outlines the approaches to modeling the protein 3D structure and probing into the spatial organization of theEscherichia coli ribosome and virus particles.     

A comparative study of the adsorption of water and methanol in zeolite BEA: a molecular simulation study

Grand canonical Monte Carlo simulations were carried out to study the equilibrium adsorption concentration of methanol and water in all-silica BEA zeolite and HBEA zeolites with different Si/Al ratios over a wide range of temperatures and loadings. These zeolites have oval-shaped channels with one side longer than the other. Water sorption into the hydrophobic BEA zeolite had a sharp transition with its sorption going from zero to near full capacity over a very small pressure range. Methanol sorption was much more gradual with respect to pressure. With the addition of hydrophilic sites for the HBEA zeolites by decreasing the Si/Al ratio, adsorption at lower pressures increased significantly for water and methanol. At higher loadings, water and methanol adsorption were found to behave in fundamentally different ways. Water structures in the zeolite channels formed hydrogen-bonded chains while maximising contact with the surfaces on the longer edges of the zeolite channels. Methanol molecules, in contrast, formed very few hydrogen bonds between themselves, with their hydroxyl groups primarily binding with surface of the shorter edge of the zeolite channels and their methyl groups located near the middle of the zeolite channels. The addition of hydrophilic groups in the HBEA zeolites strongly influenced positions of the methanol hydroxyl groups at high loadings, but did not have a significant effect on water structure.     

Immunoglobulin fusion proteins as a tool for evaluation of T-cell costimulatory molecules

Costimulatory signal(s) in addition to engagement of T cell receptor is important for optimal activation of T lymphocytes. During last decade several dozens of new costimulatory molecules, including 4-1BB, CD40, OX40, CD27, and ICOS, have been identified. It is likely that in Post-Genome Era more molecules with potential costimulatory properties will be brought to our attention. We describe here a simple and reliable strategy for evaluating the functional activities of potential costimulatory molecules using soluble fusion protein between extracellular portion of costimulatory ligand and Fc portion of mouse IgG2a.     

Template matching as a tool for annotation of tomograms of stained biological structures

In recent years, electron tomography has improved our three-dimensional (3D) insight in the structural architecture of cells and organelles. For studies that involve the 3D imaging of stained sections, manual annotation of tomographic data has been an important method to help understand the overall 3D morphology of cellular compartments. Here, we postulate that template matching can provide a tool for more objective annotation and contouring of cellular structures. Also, this technique can extract information hitherto unharvested in tomographic studies. To evaluate the performance of template matching on tomograms of stained sections, we generated several templates representing a piece of microtubule or patches of membranes of different staining-thicknesses. These templates were matched to tomograms of stained electron microscopy sections. Both microtubules and ER-Golgi membranes could be detected using this method. By matching cuboids of different thicknesses, we were able to distinguish between coated and non-coated endosomal membrane-domains. Finally, heterogeneity in staining-thickness of endosomes could be observed. Template matching can be a useful addition to existing annotation-methods, and provide additional insights in cellular architecture.     

Carbon-13 as a tool for the study of carbohydrate structures,conformations and interactions

The application of ¹³C-NMR spectroscopy to problems involving the structures and interactions of carbohydrates is described. Both ¹³C-enriched and natural abundance compounds were used and some advantages of the use of the stable isotope are described. Carbon-carbon and carbon-proton coupling constants obtained from 1-¹³ C enriched carbohydrates were employed in the assignment of their chemical shifts and to establish solution conformation. In all cases studied thus far, C-3 couples to C-1 only in the β-anomers while C-5 couples to C-1 only in the α-anomers. C-6 and C-2 always couple to C-1 in both anomeric species. The alkaline degradation of glucose [1-¹³ C] to saccharinic acids was followed by ¹³C-NMR. The conversion of glucose [1-¹³ C] to fructose-1,6-bisphosphate [1,6-¹³ C] by enzymes of the glycolytic pathway was shown as an example of the use of ¹³C-enriched carbohydrates to elucidate biochemical pathways. In a large number of glycosyl phosphates the ³¹P to H-1 and ³¹P to C-2 coupling constants demonstrate that in the preferred conformation the phosphate group lies between the O-5 and the H-1 of the pyranose ring. The influence of paramagnetic Mn²⁺ ions on the proton decoupled ¹³C-NMR spectra of uridine diphosphate N-acetylglucosamine indicates that the Mn²⁺ interacts strongly with the pyrophosphate moiety and with the carbonyl groups of the uracil and N-acetyl groups.     

In vitro selection as a powerful tool for the applied evolution of proteins and peptides

New in vitro methods for the applied evolution of protein structure and function complement conventional cellular and phage-based methods. Strategies employing the direct physical linkage of genotype and phenotype, and the compartmental association of gene and product to select desired properties are discussed, and recent useful applications are described. Engineering of antibodies and other proteins, selection from cDNA libraries, and the creation of functional protein domains from completely random starting sequences illustrate the value of the in vitro approaches. Also discussed is an emerging new direction for in vitro display technology: the self-assembly of protein arrays.     

Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function

The advantages of oriented immobilization of biologically active proteins are good steric accessibilities of active binding sites and increased stability. This not only may help to increase the production of preparative procedures but is likely to promote current knowledge about how the living cells or tissues operate. Protein inactivation starts with the unfolding of the protein molecule by the contact of water with hydrophobic clusters located on the surface of protein molecules, which results in ice-like water structure. Reduction of the nonpolar surface area by the formation of a suitable biospecifc complex or by use of carbohydrate moieties thus may stabilize proteins. This review discusses oriented immobilization of antibodies by use of immobilized protein A or G. The section about oriented immobilization of proteins by use of their suitable antibodies covers immobilization of enzymes utilizing their adsorption on suitable immunosorbents prepared using monoclonal or polyclonal antibodies, preparation of bioaffinity adsorbent for the isolation of concanavalin A and immobilization of antibodies by use of antimouse immunoglobulin G, Fc-specific (i.e. specific towards the constant region of the molecule). In the further section immobilization of antibodies and enzymes through their carbohydrate moieties is described. Oriented immobilization of proteins can be also based on the use of boronate affinity gel or immobilized metal ion affinity chromatography technique. Biotin–avidin or streptavidin techniques are mostly used methods for oriented immobilization. Site-specific attachment of proteins to the surface of solid supports can be also achieved by enzyme, e.g., subtilisin, after introduction a single cysteine residue by site-directed mutagenesis.     

Immunoblotting of a Staphylococcus aureus DNA library as a tool for isolating potential antigenic proteins

Abstract A Staphylococcus aureus DNA library was created in the lambda vector EMBL4 and expressed in Escherichia coli strain Y1090. Plaques were screened using serum from a S. aureus -infected patient. A number of positive clones that expressed proteins recognised by serum antibodies were further analysed using Western blots of total lysates from cultures infected with the lambda clones. Differences were found between patient and control sera in both the specificity and tite of anti- S. aureus antibodies. Expressed staphylococcal proteins appeared to be stable and were easily distinnguishable from E. coli proteins in the Western blot. This method may have applications in specifically selecting clones encoding antigens associated with infection and may be of potential use clinically.     

Likeness: a system for rapid retrieval of similar conformation of proteins and their spatial structures

We report a method for estimating the conformational likeness between any two proteins. Likeness is handled in terms of stochastic geometry, fuzzy logic and decision making theories. Using the method, a computer system LIKENESS was developed. The system does "real time" searching and spatial alignment of any PDB-available proteins. LIKENESS is accessible at URL: "http:@cl.sdsc.edu/".     

REMiner: a tool for unbiased mining and analysis of repetitive elements and their arrangement structures of large chromosomes

Repetitive elements (REs) constitute a substantial portion of the genomes of human and other species; however, the RE profiles (type, density, and arrangement) within the individual genomes have not been fully characterized. In this study, we developed an RE analysis tool, called REMiner, for a chromosome-wide investigation into the occurrence of individual REs and arrangement of clusters of REs, and REMiner's functional features were examined using the human chromosome Y. The algorithm implemented by REMiner focused on unbiased mining of REs in large chromosomes and data interface within a viewer. The data from the chromosome demonstrated that REMiner is an efficient tool in regard to its capacity for a large query size and the availability of a high-resolution viewer, featuring instant retrieval of alignment data and control of magnification and identity ratio. The chromosome-wide survey identified a diverse population of ordered RE arrangements, which may participate in the genome biology.     

Critical assessment of the automated AutoDock as a new docking tool for virtual screening

A major problem in virtual screening concerns the accuracy of the binding free energy between a target protein and a putative ligand. Here we report an example supporting the outperformance of the AutoDock scoring function in virtual screening in comparison to the other popular docking programs. The original AutoDock program is in itself inefficient to be used in virtual screening because the grids of interaction energy have to be calculated for each putative ligand in chemical database. However, the automation of the AutoDock program with the potential grids defined in common for all putative ligands leads to more than twofold increase in the speed of virtual database screening. The utility of the automated AutoDock in virtual screening is further demonstrated by identifying the actual inhibitors of various target enzymes in chemical databases with accuracy higher than the other docking tools including DOCK and FlexX. These results exemplify the usefulness of the automated AutoDock as a new promising tool in structure-based virtual screening.     

NMR spectroscopy as a tool for the rapid assessment of the conformation of GST-fusion proteins

Glutathione-S-transferase (GST)-fusion proteins are used extensively for structural, biochemical, and functional analyses. Although the conformation of the target protein is of critical importance, confirmation of the folded state of the target is often not undertaken or is cumbersome because of the requirement to first remove the GST tag. Here, we demonstrate that it is possible to record conventional (15)N-HSQC NMR spectra of small GST-fusion proteins and that the observed signals arise almost exclusively from the target protein. This approach constitutes a rapid and straightforward means of assessing the conformation of a GST-fusion protein without having to cleave the GST and should prove valuable, both to biochemists seeking to check the conformation of their proteins prior to functional studies and to structural biologists screening protein constructs for suitability as targets for structural studies.     

Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins

Amyloidogenesis is a characteristic feature of the 40 or so known protein deposition diseases, and accumulating evidence strongly suggests that self-association of misfolded proteins into either fibrils, protofibrils, or soluble oligomeric species is cytotoxic. The most likely mechanism for toxicity is through perturbation of membrane structure, leading to increased membrane permeability and eventual cell death. There have been a rather limited number of investigations of the interactions of amyloidogenic polypeptides and their aggregated states with membranes; these are briefly reviewed here. Amyloidogenic proteins discussed include A-beta from Alzheimer's disease, the prion protein, α-synuclein from Parkinson's disease, transthyretin (FAP, SSA amyloidosis), immunoglobulin light chains (primary (AL) amyloidosis), serum amyloid A (secondary (AA) amyloidosis), amylin or IAPP (Type 2 diabetes) and apolipoproteins. This review highlights the significant role played by fluorescence techniques in unraveling the nature of amyloid fibrils and their interactions and effects on membranes. Fluorescence spectroscopy is a valuable and versatile method for studying the complex mechanisms of protein aggregation, amyloid fibril formation and the interactions of amyloidogenic proteins with membranes. Commonly used fluorescent techniques include intrinsic and extrinsic fluorophores, fluorescent probes incorporated in the membrane, steady-state and lifetime measurements of fluorescence emission, fluorescence correlation spectroscopy, fluorescence anisotropy and polarization, fluorescence resonance energy transfer (FRET), fluorescence quenching, and fluorescence microscopy.