Orange G is a potential inhibitor of human insulin amyloid fibrillation and can be used as a probe to study mechanism of amyloid fibrillation and its inhibition.

The extracellular insoluble deposits of highly ordered cross-ß-structure-containing amyloid fibrils form the pathological basis for protein misfolding diseases. As amyloid fibrils are cytotoxic, inhibition of the process is a therapeutic strategy. Several small molecules have been identified and used as fibrillation inhibitors in the recent past. In this work, we investigate the effect of Orange G on insulin amyloid formation using fluorescence-based assays and negative-stain electron microscopy (EM). We show that Orange G effectively attenuates nucleation, thereby inhibiting amyloid fibrillation in a dose-dependent manner. Fluorescence quenching titrations of Orange G showed a reasonably strong binding affinity to native insulin. Binding isotherm measurements revealed the binding of Orange G to pre-formed insulin fibrils too, indicating that Orange G likely binds and stabilizes the mature fibrils and prevents the release of toxic oligomers which could be potential nuclei or templates for further fibrillation. Molecular docking of Orange G with native insulin and amyloid-like peptide structures were also carried out to analyse the contributing interactions and binding free energy. The findings of our study emphasize the use of Orange G as a molecular probe to identify and design inhibitors of amyloid fibrillation and to investigate the structural and toxic mechanisms underlying amyloid formation.

Comparative Immunogenicity of Bacterially Expressed Soluble Trimers and Nanoparticle Displayed Influenza Hemagglutinin Stem Immunogens.

Current influenza vaccines need to be updated annually due to mutations in the globular head of the viral surface protein, hemagglutinin (HA). To address this, vaccine candidates have been designed based on the relatively conserved HA stem domain and have shown protective efficacy in animal models. Oligomerization of the antigens either by fusion to oligomerization motifs or display on self-assembling nanoparticle scaffolds, can induce more potent immune responses compared to the corresponding monomeric antigen due to multivalent engagement of B-cells. Since nanoparticle display can increase manufacturing complexity, and often involves one or more mammalian cell expressed components, it is important to characterize and compare various display and oligomerization scaffolds. Using a structure guided approach, we successfully displayed multiple copies of a previously designed soluble, trimeric influenza stem domain immunogen, pH1HA10, on the ferritin like protein, MsDps2 (12 copies), Ferritin (24 copies) and Encapsulin (180 copies). All proteins were expressed in Escherichia coli. The nanoparticle fusion immunogens were found to be well folded and bound to the influenza stem directed broadly neutralizing antibodies with high affinity. An 8.5 Å Cryo-EM map of Msdps2-pH1HA10 confirmed the successful design of the nanoparticle fusion immunogen. Mice immunization studies with the soluble trimeric stem and nanoparticle fusion constructs revealed that all of them were immunogenic, and protected mice against homologous (A/Belgium/145-MA/2009) and heterologous (A/Puerto Rico/8/1934) challenge with 10MLD50 mouse adapted virus. Although nanoparticle display conferred a small but statistically significant improvement in protection relative to the soluble trimer in a homologous challenge, heterologous protection was similar in both nanoparticle-stem immunized and trimeric stem immunized groups. Such rapidly producible, bacterially expressed antigens and nanoparticle scaffolds are useful modalities to tackle future influenza pandemics.

Inhibition of amyloid fibril formation of lysozyme by ascorbic acid and a probable mechanism of action.

Amyloid fibrillation of proteins and polypeptides and their deposition in cells and tissues is associated with a number of pathological states collectively known as amyloid disorders. Inhibition of protein misfolding and aggregation is thus of utmost importance in the prevention and treatment of such diseases. There is a growing interest in identification of small molecules that can bind to native monomeric proteins or their partially unfolded states, thereby stabilizing them and preventing or delaying them from undergoing amyloid fibril formation. Here we report the inhibitory effect of ascorbic acid, an essential dietary component richly present in many natural food items, on the amyloid fibrillation of hen egg white lysozyme, a model protein for amyloid formation. The effect was dose dependent with more than 80% inhibition occurring even at only a five-fold molar excess of ascorbic acid. TEM images show complete absence of fibrils in the presence of ascorbic acid. From our spectroscopic and computational characterization of ascorbic acid binding to HEWL, we propose that ascorbic acid binds to the aggregation prone beta domain of HEWL, stabilizes the partially unfolded conformation and prevents further conformational changes leading to fibrillation. Hence ascorbic acid has a great therapeutic potential for amyloid disorders.

Inhibition of insulin amyloid fibrillation by Morin hydrate.

We report here the inhibition of amyloid fibrillation of human insulin in vitro by Morin hydrate, a naturally occurring small molecule. Using spectroscopic assays and transmission electron microscopy, we found that Morin hydrate effectively inhibits insulin amyloid fibrillation in a dose dependent manner with more than 80% inhibition occurring even at only a 1:1 concentration. As suggested by fluorescence spectroscopic titration studies, Morin hydrate binds to insulin with a fairly strong affinity of -26.436kJmol-1. Circular dichroism (CD) spectroscopy was used to analyse structural changes of insulin in the presence of Morin hydrate demonstrating the ability of Morin hydrate to bind with the native monomeric protein and/or its near native state, intermediate oligomeric species and amyloid fibrils. Based on computational docking and molecular dynamics study, we propose that Morin hydrate binds to residues having greater aggregation propensity and prevent structural and/or conformational changes leading to amyloid fibrillation. Morin hydrate should also bind to fibrils by hydrogen bonding and/or hydrophobic forces throughout the surface, stabilize them and inhibit the release of oligomeric species which could be nuclei or template for further fibrillation. Overall results provide an insight into the mechanism of inhibition of insulin amyloid fibrillation by Morin hydrate.

Combined in silico approaches for the identification of novel inhibitors of human islet amyloid polypeptide (hIAPP) fibrillation.

Human islet amyloid polypeptide (hIAPP) is a natively unfolded polypeptide hormone of glucose metabolism, which is co-secreted with insulin by the ß-cells of the pancreas. In patients with type 2 diabetes, IAPP forms amyloid fibrils because of diabetes-associated ß-cells dysfunction and increasing fibrillation, in turn, lead to failure of secretory function of ß-cells. This provides a target for the discovery of small organic molecules against protein aggregation diseases. However, the binding mechanism of these molecules with monomers, oligomers and fibrils to inhibit fibrillation is still an open question. In this work, ligand and structure-based in silico approaches were used to identify novel fibrillation inhibitors and/or fibril binding compounds. The best pharmacophore model was used as a 3D search query for virtual screening of a compound database to identify novel molecules having the potential to be therapeutic agents against protein aggregation diseases. Docking and molecular dynamics simulation studies were used to explore the interaction pattern and mechanism of the identified novel small molecules with predicted hIAPP structure, its aggregation prone conformation and fibril forming segments. We show that catechins with galloyl group and molecules having two to three planar apolar rings bind to hIAPP structures and fibril forming segments with greater affinity. The differences in binding affinities of different compounds against several fibril forming segments of the peptide suggest that a mixture of active compounds may be required for treatment of aggregation diseases.

Structural studies of arginine induced enhancement in the activity of T7 RNA polymerase.

Addition of arginine enhances the activity of the enzyme T7 RNA polymerase. Different methods have been employed to understand the enhancement in the light of arginine induced alteration of the tertiary structure. The increase in activity of the enzyme reaches a maximum value around a concentration of 125 mM arginine. Fluorescence, circular dichroism and dynamic light scattering studies indicate an alteration in the tertiary structure of the enzyme. Enthalpy change as a function of input concentration of arginine to a fixed concentration of the enzyme (5 µM) shows a dip at 100 mM concentration of arginine. Differential scanning calorimetric studies of the denaturation of the enzyme in absence and presence of arginine indicates arginine induced destabilization of the C-terminal domain of the enzyme. Structural alterations induced by arginine have been compared with those induced by the denaturant guanidine hydrochloride.

Biphasic association of T7 RNA polymerase and a nucleotide analogue, cibacron blue as a model to understand the role of initiating nucleotide in the mechanism of enzyme action.

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. Here we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, its effect on the function of the enzyme and the probable mode of binding of the dye. We have used difference absorption spectroscopy and isothermal titration calorimetry to show that the dye binds T7 RNAP in a biphasic manner. The first phase of the binding is characterized by inactivation of the enzyme. The second binding site overlaps with the common substrate-binding site of the enzyme. We have carried out docking experiment to map the binding site of the dye in the promoter bound protein. Competitive displacement of the dye from the high affinity site by labeled GTP and isothermal titration calorimetry of high affinity GTP bound enzyme with the dye suggests a strong correlation between the high affinity dye binding and the high affinity GTP binding in T7 RNAP reported earlier from our laboratory.

Effect of crowding agents, signal peptide, and chaperone SecB on the folding and aggregation of E. coli maltose binding protein.

SecB, a soluble cytosolic chaperone component of the Sec export pathway, binds to newly synthesized precursor proteins and prevents their premature aggregation and folding and subsequently targets them to the translocation machinery on the membrane. PreMBP, the precursor form of maltose binding protein, has a 26-residue signal sequence attached to the N-terminus of MBP and is a physiological substrate of SecB. We examine the effect of macromolecular crowding and SecB on the stability and refolding of denatured preMBP and MBP. PreMBP was less stable than MBP (DeltaT(m )= 7 +/- 0.5 K) in both crowded and uncrowded solutions. Crowding did not cause any substantial changes in the thermal stability of MBP (DeltaT(m )= 1 +/- 0.4 K) or preMBP (DeltaT(m )= 0 +/- 0.6 K), as observed in spectroscopically monitored thermal unfolding experiments. However, both MBP and preMBP were prone to aggregation while refolding under crowded conditions. In contrast to MBP aggregates, which were amorphous, preMBP aggregates form amyloid fibrils. Under uncrowded conditions, a molar excess of SecB was able to completely prevent aggregation and promote disaggregation of preformed aggregates of MBP. When a complex of the denatured protein and SecB was preformed, SecB could completely prevent aggregation and promote folding of MBP and preMBP even in crowded solution. Thus, in addition to maintaining substrates in an unfolded, export-competent conformation, SecB also suppresses the aggregation of its substrates in the crowded intracellular environment. SecB is also able to promote passive disaggregation of macroscopic aggregates of MBP in the absence of an energy source such as ATP or additional cofactors. These experiments also demonstrate that signal peptide can greatly influence protein stability and aggregation propensity.

Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies.

Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.

Design of disulfide-linked thioredoxin dimers and multimers through analysis of crystal contacts.

Disulfide bonds play an important role in protein stability and function. Here, we describe a general procedure for generating disulfide-linked dimers and multimers of proteins of known crystal structures. An algorithm was developed to predict sites in a protein compatible with intermolecular disulfide formation with neighboring molecules in the crystal lattice. A database analysis was carried out on 46 PDB coordinates to verify the general applicability of this algorithm to predict intermolecular disulfide linkages. On the basis of the predictions from this algorithm, mutants were constructed and characterized for a model protein, thioredoxin. Of the five mutants, as predicted, in solution four formed disulfide-linked dimers while one formed polymers. Thermal and chemical denaturation studies on these mutant thioredoxins showed that three of the four dimeric mutants had similar stability to wild-type thioredoxin while one had lower stability. Three of the mutant dimers crystallized readily (in four to seven days) in contrast to the wild-type protein, which is particularly difficult to crystallize and takes more than a month to form diffraction-quality crystals. In two of the three cases, the structure of the dimer was exactly as predicted by the algorithm, while in the third case the relative orientation of the monomers in the dimer was different from the predicted one. This methodology can be used to enhance protein crystallizability, modulate the oligomerization state and to produce linear chains or ordered three-dimensional protein arrays.

Thermodynamic effects of proline introduction on protein stability.

The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive.

Attempts to delineate the relative contributions of changes in hydrophobicity and packing to changes in stability of ribonuclease S mutants.

While the hydrophobic driving force is thought to be a major contributor to protein stability, it is difficult to experimentally dissect out its contribution to the overall free energy of folding. We have made large to small substitutions of buried hydrophobic residues at positions 8 and 13 in the peptide/protein complex, RNase-S, and have characterized the structures by X-ray crystallography. The thermodynamics of association of these mutant S peptides with S protein was measured in the presence of different concentrations of methanol and ethanol. The reduction in the strength of the hydrophobic driving force in the presence of these organic solvents was estimated from surface-tension data as well as from the dependence of the DeltaC(p) of protein/peptide binding on the alcohol concentration. The data indicated a decrease in the strength of the hydrophobic driving force of about 30-40% over a 0-30% range of the alcohol concentration. We observe that large to small substitutions destabilize the protein. However, the amount of destabilization, relative to the wild type, is independent of the alcohol concentration over the range of alcohol concentrations studied. The data clearly indicate that decreased stability of the mutants is primarily due to the loss of packing interactions rather than a reduced hydrophobic driving force and suggest a value of the hydrophobic driving force of less than 18 cal mol(-)(1) A(2).

Association of fluorescent probes 1-anilinonaphthalene-8-sulfonate and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid with T7 RNA polymerase.

T7 RNA polymerase is an enzyme that carries out transcription using DNA as the template and ribonucleotides as the substrates. Here we report the association of the polymerase with 1-anilinonaphthalene-8-sulfonate (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS), which are two fluorescent hydrophobic probes that are frequently used to study structural perturbations in proteins and intermediate states of proteins during folding and unfolding. Our results from the fluorescence titration data show that these two molecules bind to the enzyme with dissociation constants on the micromolar order. The results from the tryptic digestion of the enzyme in the absence and presence of the probes show that they inhibit the rate of tryptic digestion. Circular dichroism spectroscopic studies of the protein in the near UV region indicate that both probes induce tertiary structural changes in the polymerase. There is also a probe (ANS or bis-ANS) induced inhibition of the enzymatic activity. All these results are attributed to association of the probes with the enzyme, leading to an alteration in the conformation of T7 RNA polymerase. This limits the use of these extrinisic probes to the study of the folding properties of the enzyme.