Spectroscopic and computational studies on the binding interaction between gallic acid and Pin1.

Gallic acid (GA) is a natural ingredient in functional foods, which has various health-promoting and antitumour effects. Peptidyl-prolyl cis/trans isomerase Pin1 plays an important role in preventing the development of some malignant tumours. However, whether there was an interaction between Pin1 and GA remains unknown. In this work, the binding information of GA and Pin1 was investigated systematically using multiple spectral and computational methods. GA bound to Pin1 directly with moderate binding affinity in the order of 104 mol/L, therefore decreasing the activity of Pin1. Also, the binding process of GA to Pin1 was driven through weak van der Waals forces, hydrogen bonds, and electrostatic forces. In addition, the important residues Lys63, Arg68, and Arg69 played a significant role in maintaining the binding stability between Pin1 and GA. Interestingly, GA reduced the activity of Pin1 by affecting its conformational characteristics. Our present work showed that GA binds to Pin1 and inhibits its activity, affecting its structural and functional properties, which may contribute to the therapy of Pin1-related diseases.

Genomic distribution and possible functional roles of putative G-quadruplex motifs in two subspecies of Oryza sativa.

G-quadruplex is a stable, four-stranded DNA or RNA structure formed from guanine-rich regions and implicated in telomere maintenance, replication, gene regulation at transcription level or translation level, etc. Based on bioinformatics methods, we analyzed different putative G-quadruplex motifs (PGQMs) patterns in various genomic regions of two subspecies (indica and japonica) of Oryza sativa and the whole genomes of other 8 species. In total, in the 10 species we discussed, the PGQMs densities in monocots were higher than dicots. 40,483 and 31,795 PGQMs were identified with a density of 108.46 and 84.89 PGQMs/Mb, respectively, in japonica and indica genomes, 10,655 and 5420 loci were found to contain at least one PGQM in their gene bodies (with a percentage of 19% and 14%) indicating a wide distribution of G-quadruplex motifs in O. sativa genome. They preferred to locate in transcription start sites proximal regions and 5'-UTR with relative high enrichment. This phenomenon supports the hypothesis that PGQMs are involved in gene transcription and translation. In addition, we analyzed the distribution of different loop length in G-quadruplex and found the density of long loop PGQMs was less than short loop in indica's intron but it was similar in japonica. Meanwhile, we focused on the loci with PGQMs and conducted gene ontology (GO) analysis of them. As a result, many GO terms were identified and significantly correlated with the loci containing at least one PGQM. The GO analysis in the two subspecies of rice may be helpful for elucidating the functional roles of G-quadruplexes.

The structural and functional role of the three tryptophan residues in Pin1.

Pin1, the only known isomerase catalyzing phosphorylated pSer/pThr-Pro motifs in proteins, plays unique roles in human diseases notably cancers and Alzheimer's disease. Herein, site-directed mutagenesis was employed to construct the tryptophan mutants of Pin1, including W11L, W34L, and W73L. Spectral methodologies, activity measurement, and proteinase resistance analysis were used to investigate the structural and functional role of the tryptophan residues in Pin1. In general, W11 and W34 are essential to the structure and the function of Pin1, because their mutations influence the structure of WW domain of Pin1, potentially attenuate the binding affinity of Pin1 to substrates, and thus inhibit the enzymatic activity of Pin1. Particularly, W11 mutation results in significantly varied structural features of Pin1 as revealed by fluorescence and circular dichroism (CD) spectroscopies, and decreases the enzymatic activity, thermal stability, and proteinase resistance of Pin1, all of which give an explanation for the high conservation of W11 in vivo. The synchronous fluorescence spectra indicate that W11 and W34 mutations possibly block their energy transfer to Y23 or Y24, suggesting the structural rearrangement in WW domain. By contrast, W73 is of minor importance for the structure and the function of Pin1, because the parameters of W73L observed in several experiments are very similar to wild-type Pin1. These observations are very beneficial for further understanding the structural and functional characteristics of Pin1 and for unveiling the pathogenesis of Pin1-related diseases especially those caused by tryptophan mutations.

Soluble expression of Spinach psbC gene in Escherichia coli and in vitro reconstitution of CP43 coupled with chlorophyll a only.

CP43 is a chlorophyll a (Chl a) and ß-carotene (ß-Car) binding protein encoded by psbC gene. In this study, psbC gene isolated from Spinach was expressed in Escherichia coli in soluble state. After lysis of the cells, the apoproteins purified by nickel affinity chromatography were examined by SDS-PAGE and Western-blot. Next, reconstitution experiment was carried out in vitro and the formation of stable pigment-protein complex was analyzed by partially denaturing electrophoresis. After purifying reconstituted CP43 (rCP43) from free pigments (FPs) by sucrose gradient ultracentrifugation and subsequently ion exchange chromatography (IEC), the eluate was analyzed by partially denaturing electrophoresis to confirm stability of the reconstructed complex. Finally, analyses of spectroscopic character of the eluate revealed that in vitro reconstitution was achieved and FPs were completely removed from the pigment-protein complex. Comparison between the absorption spectra of the rCP43 and native CP43 (nCP43) showed the lack of peaks between 450 and 500 nm, illustrating that the ß-Car was stripped off rCP43. In brief, it is feasible to obtain a reconstituted protein binding Chl a only, indicating that the occupancy of the ß-Car site has small impact on the stabilization of CP43. However, ß-Car shows strong interaction with Chl a, inducing the hyperchromic effect in blue region of spectrum and the blue shift of the 438.5 nm and 673.5 nm absorption band to 437 nm and 671 nm respectively. To some extent, our research is suggestive that ß-Car, coupled loosely with CP43, contributes to the precise orientation of Chl a in vivo.

Insight into the structural stability of wild type and mutants of the tobacco etch virus protease with molecular dynamics simulations.

The efficiency and high specificity of tobacco etch virus protease (TEVp) has made it widely used for cleavage of recombinant fusion proteins. However, TEVp suffers from a few intrinsic defects such as self-cleavage, poorly expressed in E. coli and less soluble. So some mutants were designed to improve it, such as S219V, T17S/N68D/I77V and L56V/S135G etc. MD simulations for the WT TEVp and its mutants were performed to explore the underlying dynamic effects of mutations on TEVp. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of TEVp, including the elongation of ß-sheet, conversion of loop to helix and the flexibility of active core. Our present study indicates that the three mutants for TEVp can change their secondary structure and tend to form more helixes and sheets to improve stability. The study also helps us to understand the effects of some mutations on TEVp, provides us insights into the change of them at the atomic level and gives a potential rational method to design an improved protein.

The acidic pH-induced structural changes in Pin1 as revealed by spectral methodologies.

Pin1 is closely associated with the pathogenesis of cancers and Alzheimer's disease (AD). Previously, we have shown the characteristics of the thermal denaturation of Pin1. Herein, the acid-induced denaturation of Pin1 was determined by means of fluorescence emission, synchronous fluorescence, far-UV CD, ANS fluorescence and RLS spectroscopies. The fluorescence emission spectra and the synchronous fluorescence spectra suggested the partially reversible unfolding (approximately from pH 7.0 to 4.0) and refolding (approximately from pH 4.0 to 1.0) of the structures around the chromophores in Pin1, apparently with an intermediate state at about pH 4.0-4.5. The far-UV CD spectra indicated that acidic pH (below pH 4.0) induced the structural transition from α-helix and random coils to ß-sheet in Pin1. The ANS fluorescence and the RLS spectra further suggested the exposure of the hydrophobic side-chains of Pin1 and the aggregation of it especially below pH 2.3, and the aggregation possibly resulted in the formation of extra intermolecular ß-sheet. The present work primarily shows that acidic pH can induce kinds of irreversible structural changes in Pin1, such as the exposure of the hydrophobic side-chains, the transition from α-helix to ß-sheet and the aggregation of Pin1, and also explains why Pin1 loses most of its activity below pH 5.0. The results emphasize the important role of decreased pH in the pathogenesis of some Pin1-related diseases, and support the therapeutic approach for them by targeting acidosis and modifying the intracellular pH gradients.

Spectroscopic studies on the irreversible heat-induced structural transition of Pin1.

Previously, the mechanism of the thermal unfolding of Pin1 (on-line measurements) was studied, revealing that Pin1 has a relatively high thermal stability. However, it is still questionable whether the unfolding of Pin1 is reversible. In the present work, intrinsic tryptophan fluorescence, ANS fluorescence, RLS, FTIR and CD spectroscopies are used to evaluate the reversibility of the thermal unfolding of Pin1. Intrinsic tryptophan fluorescence studies indicate that structural changes around tryptophan motifs in Pin1 are possibly reversible after heat treatment (even above 98°C), for no significant change in the intensity or λ(max) of the spectra was observed. ANS fluorescence measurements indicate the irreversible exposure of the hydrophobic clusters in Pin1 after heat treatment at 98°C, with increase in the fluorescence intensity and blue shift in λmax. Also, RLS signals of the Pin1-ANS system increased after heat treatment, possibly implying both the unfolding and the aggregation of Pin1. In addition, FTIR and CD results confirmed the irreversible unfolding of the secondary structure in Pin1 after heat treatment above 90°C, showing decreases in both α-helix and ß-sheet. In summary, the present work mainly suggests that heat treatment, especially above 90°C, has an important impact on the structural stability of Pin1, and the structural unfolding induced by heat was proved to be irreversible.