Pushpa Mishra

Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters

Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.

NMR Insights into Folding and Self-Association of Plasmodium falciparum P2

The eukaryotic 60S-ribosomal stalk is composed of acidic ribosomal proteins (P1 and P2) and neutral protein P0, which are thought to be associated as a pentameric structure, [2P1, 2P2, P0]. Plasmodium falciparum P2 (PfP2) appears to play additional non-ribosomal functions associated with its tendency for homo-oligomerization. Recombinant bacterially expressed PfP2 protein also undergoes self-association, as shown by SDS-PAGE analysis and light scattering studies. Secondary structure prediction algorithms predict the native PfP2 protein to be largely helical and this is corroborated by circular dichroism investigation. The 1H-15N HSQC spectrum of native P2 showed only 43 cross peaks compared to the expected 138. The observed peaks were found to belong to the C-terminal region, suggesting that this segment is flexible and solvent exposed. In 9 M urea denaturing conditions the chain exhibited mostly non-native β structural propensity. 15N Relaxation data for the denatured state indicated substantial variation in ms-µs time scale motion along the chain. Average area buried upon folding (AABUF) calculations on the monomer enabled identification of hydrophobic patches along the sequence. Interestingly, the segments of slower motion in the denatured state coincided with these hydrophobic patches, suggesting that in the denatured state the monomeric chain undergoes transient hydrophobic collapse. The implications of these results for the folding mechanism and self-association of PfP2 are discussed.

Reduced dimensionality 3D HNCAN for unambiguous HN, CA and N assignment in proteins.

We present here an improvisation of HNN (Panchal, Bhavesh et al., 2001) called RD 3D HNCAN for backbone (HN, CA and (15)N) assignment in both folded and unfolded proteins. This is a reduced dimensionality experiment which employs CA chemical shifts to improve dispersion. Distinct positive and negative peak patterns of various triplet segments along the polypeptide chain observed in HNN are retained and these provide start and check points for the sequential walk. Because of co-incrementing of CA and (15)N, peaks along one of the dimensions appear at sums and differences of the CA and (15)N chemical shifts. This changes the backbone assignment protocol slightly and we present this in explicit detail. The performance of the experiment has been demonstrated using Ubiquitin and Plasmodium falciparum P2 proteins. The experiment is particularly valuable when two neighboring amino acid residues have nearly identical backbone (15)N chemical shifts.

Residue level description of In vivo self-association of Plasmodium falciparum P2

Plasmodium falciparum P2 (PfP2) is a ribosomal stalk protein. It also performs extra ribosomal novel functions that seem to be associated with homo oligomerization . Previous in vitro studies have demonstrated that the protein has a high tendency to self-associate predominantly into an 8-mer. In vitro Heteronuclear Single Quantum Coherence (HSQC) of the pure recombinant protein (rPfP2) and its in-cell (Escherichia coli) HSQC spectrum has very similar features, indicating that the protein intrinsically, both inside the cell and under in vitro conditions, has similar aggregation tendencies. In view of this, we have characterized here the folding and concomitant self-association of rPfP2, using an in vitro dissociation–association strategy. We observed that the residue stretch, (Met31-Leu44) of the rPfP2, mapping to Met1-Leu14 of PfP2 protein acts as a nucleation site for helix formation and subsequent self-association. Further association appears to be driven by hydrophobic and complimentary electrostatic charge interactions on the surfaces formed. One stretch of rPfP2, (Ile97-Ala116), always remains floppy, and this may serve as “hinge” for protein segmental motions. Based on these, we have proposed a possible model for rPfP2 self-association into an 8-mer.

EWGWS insert in Plasmodium falciparum ookinete surface enolase is involved in binding of PWWP containing peptides: Implications to mosquito midgut invasion by the parasite.

There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host-pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids ((104)EWGWS(108)) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking (104)EWGWS(108), (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed.

Ribosomal Protein P2 from apicomplexan parasite Toxoplasma gondii is intrinsically a molten globule

Toxoplasma gondii is an apicomplexan parasite, which causes toxoplasmosis. Toxoplasma P2 (TgP2) is a ribosomal protein and exists as supramolecular assembly with other proteins in the ribosome. It is also shown that TgP2 is involved in some extra ribosomal functions. However, till date the protein has evaded structural characterization by any of the known techniques. In this background, we report here a systematic study using a variety of biophysical techniques and NMR, under different conditions of pH and temperature, and deduce that TgP2 consists of only helices and unstructured regions, is a monomer at low pH but forms multimers at higher pH, and has intrinsically a molten globule structure. The C-terminal half is flexible and the helices are concentrated in the N-terminal half of the chain. The dynamism inherent to the molten globule structure may have functional implications for its extra-ribosomal functions. which is contrast to that of human P2.

The C-terminal domain of eukaryotic acidic ribosomal P2 proteins is intrinsically disordered with conserved structural propensities.

The P2 protein (equivalent of L7/L12 in prokaryotes), a member of the ribosomal stalk in eukaryotes, is highly conserved, particularly its C-terminal domain. In order to understand the sequence-structure-function relationships in eukaryotic C-terminal stretches, about which nothing is known at the moment, we have investigated here, the structural characteristics of these domains of P2 proteins from three different species, namely, human, Plasmodium falciparum, and Toxoplasma gondii; the sequence homology among these is 70% although sequence identity is only 36%. About 50 amino acids of the C-terminal domains of P2 from the three species were expressed and purified. Gel filtration studies indicated peaks for both monomer and oligomer at milimolar concentrations and also suggested monomer-multimer equilibrium. Circular Dichroism showed that this domain does not have stable secondary structures. (1)H-(15)N HSQC spectra in every case showed one set of requisite number of peaks as per the sequence. This indicated that there is rapid multimer-monomer equilibrium in solution and the observed peaks which originate from the monomer reflect average chemical shifts. The spectral dispersion in all the cases is narrow, although there are noticeable differences in the three proteins. Detailed NMR investigations revealed that this protein domain is intrinsically disordered although there are short segments with preferred secondary structural propensities at similar places along the sequence. This may suggest that the sequence is selected in evolution to impart disorder, and thereby accord conformational adaptability.

152 Molten globule behavior of apicomplexan protein P2 from Plasmodium falciparum and Toxoplasma gondii.

and theoretical studies, the manner by which a protein controls the thermodynamics and kinetics of proton-transfer through the reaction cycle to accomplish proton pumping is still not well understood. Within bR there are several key sites that undergo protonation changes throughout the photocycle. The mechanism that governs the long-range proton translocation between the central (CC-the retinal Schiff base and D85) and exit clusters (EC-E194 and E204) and subsequent release to the low pH side of the membrane remains elusive. To further advance our understanding of the proton pumping mechanism of bR, a systematic study using molecular dynamics (MD) simulations-based approach in conjunction with pKa calculations with MCCE method (Song, Mao, & Gunner, 2009) were performed for seven bR models. Quantum mechanical (QM) intrinsic reaction coordinate (IRC) calculations were carried out for proton-transfer in guanidinium-water model systems. The goal is to evaluate the connectivity of the hydrogenbonding network between the CC and EC in different stages of the bR photocycle. The key finding includes:

Molten globule nature of Plasmodium falciparum P2 homo-tetramer

The P2 protein in Plasmodium falciparum has a high tendency to oligomerize, which seems to drive many of its non-ribosomal functions. During nuclear division of the parasite inside RBC, P2 translocates to the RBC surface as a tetramer. From a systematic study using variety of biophysical techniques, NMR spectral characteristics and relaxation dispersion measurements under different conditions of pH and/or urea concentrations, we deduce that (i) PfP2, an almost entirely helical protein, forms a molten globule monomer at low pH, (ii) at physiological pH, and at micro-molar concentrations, PfP2 is a stable tetramer wherein two dimmers associate sideways with close packing of helices at the interface, and (iii) the molten globule characteristic of the monomer is preserved in the tetramer. This dynamism in the structure of PfP2 may have functional implications since it is known that different kinds of oligomers are transiently formed in the parasite.