•Pin1 is governed by two types of quenching mechanisms.•The higher concentration of EGCG could make the secondary structure of Pin1 change.•Hydrogen bonds, electrostatic interaction and van der Waals interaction played the major role in the binding process.•EGCG was bound to the Glu12, Lys13, Arg14, Met15 and Arg17 in WW domain,•EGCG could interact with Arg69, Asp112, Cys113 and Ser114 in PPIase domainThe binding of epigallocatechin-3-gallate (EGCG) to wild type Pin1 in solution was studied by spectroscopic methods and molecular dynamics simulations in this research to explore the binding mode and inhibition mechanism. The binding constants and number of binding sites per Pin1 for EGCG were calculated through the Stern-Volmer equation. The values of binding free energy and thermodynamic parameters were calculated and indicated that hydrogen bonds, electrostatic interaction and Van der Waals interaction played the major role in the binding process. The alterations of Pin1 secondary structure in the presence of EGCG were confirmed by far-UV circular dichroism spectra. The binding model at atomic-level revealed that EGCG was bound to the Glu12, Lys13, Arg14, Met15 and Arg17 in WW domain. Furthermore, EGCG could also interact with Arg69, Asp112, Cys113 and Ser114 in PPIase domain.

•The thermal-induced unfolding of recombinant TEVp had been investigated.•Thermal denaturation of TEVp involved a three-state transition.•The transition from the native state to the stable intermediate was reversible.•The transition from intermediate to denatured state was irreversible.•Protein aggregations were found at higher temperature.Tobacco etch virus protease (TEVp) is widely used to remove fusion tags from recombinant proteins because of its high and unique specificity. This work describes the conformational and the thermodynamic properties in the unfolding/refolding process of TEVp3M (three-point mutant: L56V/S135G/S219V) induced by temperature. With temperature increasing from 20 to 100 °C, the CD spectra showed a transition trend from α-helix to β-sheet, and the fluorescence emission, synchronous fluorescence, ANS and RLS spectroscopy consistently revealed that the temperature-induced unfolding process behaved in a three-state manner, for there was a relatively stable intermediate state observed around 50 °C. The reversibility of thermal unfolding of TEVp3M further showed that the transition from the native to the intermediate state was reversible (below 50 °C), however the transition from the intermediate to the unfolded state was irreversible (above 60 °C). Moreover, aggregates were observed above 60 °C as revealed by SDS–PAGE, Thioflavin-T fluorescence and Congo red absorbance.

•The tryptophan mutations resulted in decreased activity of Pin1.•W11 and W34 mutations caused the multiple alterations of Pin1 structure.•W11 and W34 mutations may attenuate the binding affinity of Pin1 to substrates.•W11 mutation also resulted in the decreased resistance of Pin1 to proteinase.•W73 had minor influence on the structure and the function of 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.

The interaction of ginkgolic acid (15:1, GA) with human serum albumin (HSA) was investigated by FT–IR, CD and fluorescence spectroscopic methods as well as molecular modeling. FT–IR and CD spectroscopic showed that complexation with the drug alters the protein’s conformation by a major reduction of α-helix from 54 % (free HSA) to 46–31 % (drug–complex), inducing a partial protein destabilization. Fluorescence emission spectra demonstrated that the fluorescence quenching of HSA by GA was by a static quenching process with binding constants on the order of 105 L·mol−1. The thermodynamic parameters (ΔH = −28.26 kJ·mol−1, ΔS = 11.55 J·mol−1·K−1) indicate that hydrophobic forces play a leading role in the formation of the GA–HSA complex. The ratio of GA and HSA in the complex is 1:1 and the binding distance between them was calculated as 2.2 nm based on the Förster theory, which indicates that the energy transfer from the tryptophan residue in HSA to GA occurs with high probability. On the other hand, molecular docking studies reveal that GA binds to Site II of HSA (sub-domain IIIA), and it also shows that several amino acids participate in drug–protein complexation, which is stabilized by H-bonding.

•Al(III) quenches the intrinsic fluorescence of W11 and W34 in Pin1 at pH 4.0.•Al(III) induces the increased β-sheet and the decreased α-helix in Pin1 at pH 4.0.•Al(III) induces the increased thermal stability of Pin1 at pH 4.0.•Al(III) significantly inhibits the PPIase activity of Pin1.The enzyme peptidyl-prolyl cis-trans isomerase (Pin1) may play an important role in preventing the development of Alzheimer's disease (AD). The structural and functional stability of Pin1 is extremely important. Previously, we have determined the stability of Pin1 under stressed conditions, such as thermal treatment and acidic-pH. Considering that aluminum (Al(III)) is well known for its potential neurotoxicity in the pathogenesis of AD, we examined whether Al(III) affects the structure and function of Pin1, by means of a PPIase activity assay, intrinsic fluorescence, circular dichroism (CD) spectroscopy, FTIR, and differential scanning calorimetry (DSC). The intrinsic tryptophan fluorescence measurements mainly show that Al(III) may bind to the clusters nearby W11 and W34 in the WW domain of Pin1, quenching the intrinsic fluorescence of the two tryptophan residues, which possibly results in the decreased binding affinity of Pin1 to substrates. The secondary structural analysis as revealed by FTIR and CD measurements indicate that Al(III) induces the increase in β-sheet and the decrease in α-helix in Pin1. Furthermore, the changes of the thermodynamic parameters for Pin1 as monitored by DSC confirm that the thermal stability of Pin1 significantly increases in the presence of Al(III). The Al(III)-induced structural changes of Pin1 result in a sharp decrease of the PPIase activity of Pin1. To some extent, our research is suggestive that Al(III) may inhibit the isomerization activity of Pin1 in vivo, which may contribute to the pathogenesis of AD.Aluminum(III) interferes with the structure and the activity of the peptidyl-prolyl cis-trans isomerase Pin1, potentially contributing to Pin1-related diseases.

•TEVp is a unique enzyme with the specific enzymatic activity.•Hg2+ induced the increased hydrophobicity around the chromophores in TEVp.•Hg2+ quenched the fluorescence of TEVp mainly in a static process.•TEVp and Hg2+ possibly formed a complex with the molar ratio of 1:1.•Hg2+ may bind to the residues His46 and Cys151 and increase β-sheet in TEVp.Tobacco etch virus protease (TEVp) has specific and highly-conserved enzymatic activity, and is frequently applied in the fields of biology and biotechnology. Hg2+ can be utilized to purify target proteins in metal-chelate affinity chromatography. However, because Hg2+ has special biological toxity, it may endanger the stability of TEVp and hinder the utilization of TEVp in the related research. We determined the effects of Hg2+ on the structure of TEVp by means of intrinsic fluorescence, synchronous fluorescence, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopies. The fluorescence emission and the synchronous fluorescence spectra suggested that Hg2+ induced the increased hydrophobicity around both of the tryptophan and the tyrosine residues in TEVp. Hg2+ mainly quenched the fluorescence of TEVp in a static process, with the possible formation of a complex (approximate molar ratio of 1:1), and the electrostatic force was the main binding force between TEVp and Hg2+. The FTIR and CD spectra suggested that Hg2+ induced the increased content of β-sheet in TEVp, indicating that Hg2+ may bind to the catalytic residues His46 and Cys151 in TEVp and promote TEVp to form a more compact structure. The Hg2+-induced structural changes of TEVp may potentially inhibit the catalytic activity of TEVp. These findings are beneficial for the intensive understanding of the factors that influence the stability of TEVp, and are helpful for the efficient and effective application of TEVp in the fields of biology and biological engineering.

Plastid-to-nucleus retrograde signaling coordinates the expression of nuclear photosynthetic genes with the developmental and functional state of the plastid. These signals are essential not only for coordinating the expression of photosynthetic genes both in the plastome and nuclear genome, but also for plants to respond optimally to environmental stress. In the present study, we found that the expression of the nuclear genes that encode plastid and non-plastid photosynthesis-related proteins was still maintained or slightly higher in cr3529, a chlorophyll deficient mutant of oilseed rape that possesses an arrested development of chloroplasts, suggesting that the expression of photosynthesis-related nuclear genes was uncoupled from the normal dependence on the developmental state of the chloroplast in cr3529. When the development of the plastid in cr3529 and the wild type was completely inhibited by lincomycin, much higher expression of photosynthesis-related nuclear genes was observed in cr3529, suggesting that the genomes uncoupled (gun) phenotype of cr3529 is even more apparent than under normal growth conditions. Lincomycin treatment also derepressed the expression of plastid genes in cr3529. The determination of porphyrin flux through Mg-chelatase showed that the content of protoporphyrin IX and Mg-protoporphyrin decreased in cr3529. The obvious gun phenotype of cr3529 under normal growth conditions and the pattern of tetrapyrrole metabolism in cr3529 suggest that it is a new gun mutant that could be used to study the regulation of the expression of nuclear and plastid genes by plastid-to-nucleus retrograde signaling under more physiological conditions and the mechanism of plant stress responses mediated by plastid signals.

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 β-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 β-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 β-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.Graphical abstractHighlights► We studied the acid-induced denaturation of Pin1 by spectral methodologies. ► Acidic pH induces the partially reversible unfolding and refolding of Pin1. ► Acidic pH induces the structural transition from α-helix to β-sheet in Pin1. ► Acidic pH induces the aggregation of the hydrophobic side-chains of Pin1. ► The aggregation of Pin1 possibly results in the intermolecular β-sheet.

TAp63γ plays as an important tumor suppressor gene protecting from cancer development, especially in p53-deficient cancer cells under stresses. Here, we investigated the effects of heat treatment on the functional and structural stabilities of TAp63γ by means of the electrophoretic mobility shift assay, intrinsic tryptophan fluorescence, exogenous ANS fluorescence, and CD spectroscopies. The electrophoretic mobility shift assay result showed that the DNA binding activity of GST-TAp63γ decreased above 55 °C. The intrinsic fluorescence spectra indicated an increase of the hydrophobicity and a decrease of the polarity in the microenvironments around the tyrosine and tryptophan residues. The ANS fluorescence spectra suggested that the hydrophobic pockets in TAp63γ gradually unfolded below 50 °C. The above results indicated that TAp63γ partially unfolded at 55 °C, while the CD result showed that TAp63γ still processed a pronounced secondary structure at the same temperature, suggesting that heat treatment possibly induced the molten globule state of TAp63γ, which was an intermediate state between the native and denatured protein. Taken together, TAp63γ is a relatively unstable protein, but it has higher activity than p53 at about 50 °C. The presented work also implies that TAp63γ may play an important role in stressed microenvironments especially when p53 is deficient.Highlights► The DNA binding activity of GST-TAp63γ decreased above 55 °C. ► The polarity around tyrosines and tryptophans in TAp63γ decreased after heating. ► The hydrophobic pockets in TAp63γ gradually unfolded below 50 °C. ► Heat treatment possibly induced the molten globule state of TAp63γ at about 55 °C.

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 λmax 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 β-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.

In Arabidopsis thaliana, STN7 kinase is required for phosphorylation of LHCII and for state transitions. In this paper, a hydrophilic polypeptide, derived from the amino acid sequence of STN7, was conjugated to a carrier protein, bovine serum albumin (BSA), to obtain the polyclonal antibody. Immunogenicity and specificity of the polyclonal antibody were evaluated by agar gel immunodiffusion (AGID) test and Western blot analysis. The results show that besides the phosphorylation of LHCII proteins, also the expression of STN7 was regulated by temperature conditions. In addition, the change tendency of LHCII proteins phosphorylation was not only coherent with expression of STN7 with respect to increasing temperature, but also closely related to state transitions. These results would provide useful information for studying regulatory mechanism of LHCII proteins phosphorylation and expression of STN7.

Essential hypertension is a major risk factor for cardiovascular morbidity and mortality, and the early-diagnosis is very important for the prevention of essential hypertension. Previously, we found that Pin1, the only known enzyme isomerizing pSer/pThr-Pro motifs in proteins, may gradually become inactive under conditions of stress such as intracellular acidification and fever. Interestingly, essential hypertension and the dysfunction of Pin1 often synchronously occur with the increasing age. Recent evidence indicates that Pin1 primarily increases the activity of endothelial nitric oxide synthase (eNOS) and the production of nitric oxide (NO) in multiple ways, significantly promoting the relaxation response of blood vessels and preventing the elevation of blood pressure. Further, the inhibition of Pin1 results in significantly increased blood pressure in rats. So, we hypothesized and evaluated the potential of Pin1 to be a new early-diagnostic biomarker as well as a therapeutic drug for essential hypertension. The unique activity of Pin1 and some epidemiological and experimental data evidence that the decreased activity of Pin1 may be closely associated with the development of essential hypertension. The factors that may impact the activity of Pin1 and correlate with the risk of essential hypertension were also discussed. These findings indicate that Pin1 plays a key and permanent role in efficiently preventing the development of essential hypertension, and that Pin1 may be a promising early-diagnostic biomarker as well as an effective therapeutic drug for the early-diagnosis, prevention, and treatment of essential hypertension, potentially decreasing the risk of cardiovascular morbidity and mortality.

•Ningnanmycin possesses an inhibition effect of TMV-CP in vitro polymerization.•Ningnanmycin induces accumulation of a set of acidic PR proteins and the expression of the NPR1and Jaz3.•Ningnanmycin could induce tobacco systemic resistance against TMV via activation of multiple plant defense pathways.Ningnanmycin (NNM) is an antiviral agent firstly isolated from Strepcomces noursei var·xichangensisn. Studies have shown that NNM promotes PAL, POD and SOD activity and possesses antiviral activity against tobacco mosaic virus (TMV). In this study, our results demonstrated that NNM inhibited the polymerization process of TMV coat protein (TMV-CP) in vitro and promoted the systemic accumulation of pathogenesis-related proteins (PRs), which are the markers of systemic acquired resistance (SAR). An non-expressor, pathogenesis-related genes 1 (NPR1) that regulates SAR and induces systemic resistance (ISR), increased. In addition, the Jaz3 expression increase showed that NNM also induced ISR. Based on the results of this work and earlier reports, it is suggesting that NNM induces tobacco systemic resistance against TMV via activating multiple plant defense signaling pathways.Download full-size image

Protein ubiquitination and the subsequent degradation are important means by which aberrant proteins are removed from cells, a key requirement for long-term survival. In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase. Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function. We demonstrate here that the decrease of ubiquitinated proteins in sch9Δ cells in log phase is not caused by changes in ubiquitin expression, proteasome activity, or autophagy, but by enhanced expression of stress response factors and a decreased level of oxidative stress. Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity.