Intrinsic Protein Research Articles

Intrinsic protein disorder uncouples affinity from binding specificity.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins often function by molecular recognition, in which they undergo induced folding. Based on prior generalizations, the idea prevails in the IDP field that due to the entropic penalty of induced folding, the major functional advantage associated with this binding mode is “uncoupling” specificity from binding strength. Nevertheless, both weaker binding and high specificity of IDPs/IDRs rest on limited experimental observations, making these assumptions more speculations than evidence‐supported facts. The issue is also complicated by the rather vague concept of specificity that lacks an exact measure, such as the K d for binding strength. We addressed these issues by creating and analyzing a comprehensive dataset of well‐characterized ID/globular protein complexes, for which both the atomic structure of the complex and free energy (ΔG, K d) of interaction is known. Through this analysis, we provide evidence that the affinity distributions of IDP/globular and globular/globular complexes show different trends, whereas specificity does not connote to weaker binding strength of IDPs/IDRs. Furthermore, protein disorder extends the spectrum in the direction of very weak interactions, which may have important regulatory consequences and suggest that, in a biological sense, strict correlation of specificity and binding strength are uncoupled by structural disorder.

The Myth of The Annular Lipids.

In the early 1970s, the existence of a "lipid annulus" stably surrounding the individual intrinsic protein molecules was proposed by several authors. They referred to a number of lipid molecules in slow exchange with the bulk lipid in the bilayer, i.e., more or less protein-bound, and more ordered than the bulk lipid. The annular lipids would control enzyme activity. This idea was uncritically accepted by most scientists working with intrinsic membrane proteins at the time, so that the idea operated like a myth in the field. However, in the following decade, hard spectroscopic and biochemical evidence showed that the proposed annular lipids were not immobilized for a sufficiently long time to influence enzyme or transporter activity, nor were they ordered by the protein. Surprisingly, forty years later, the myth survives, and the term 'annular lipid' is still in use, in a different, but even more illogical sense.

Overexpression of an aquaporin gene EsPIP1;4 enhances abiotic stress tolerance and promotes flowering in Arabidopsis thaliana

Aquaporins are water channel proteins that play an essential role in plant growth and development. Despite extensive functional characterization of aquaporins in model plants such as Arabidopsis, their contributions to abiotic stress tolerance in non-model plants are still poorly understood. As a close relative of Arabidopsis thaliana, Eutrema salsugineum is an excellent model for studying salt tolerance. Here, we identified and functionally characterized EsPIP1;4, a gene encoding a plasma membrane intrinsic protein (PIP) aquaporin in E. salsugineum. Overexpression of EsPIP1;4 in Arabidopsis improved seed germination and root growth of transgenic plants under abiotic stress, which was accompanied by an increase in proline accumulation, reduction in MDA, and decrease in the rate of ion leakage. Under abiotic stress, transgenic plants overexpressing EsPIP1;4 also showed increased antioxidant enzyme activity, and enhanced K+/Na+ ratio compared to control plants. Furthermore, overexpression of EsPIP1;4 promoted flowering by regulating genes in multiple flowering pathways. Together, our results demonstrated that an aquaporin from E. salsugineum improves abiotic stress tolerance and promotes flowering.

Modulated photophysical properties and sequestration of potent anti-acetylcholinesterase active coumarinyl dyes in human serum albumin

Multi-pronged spectroscopic techniques were employed to provide quantitative inputs on the modulated photophysics of nine coumarinyl derivatives in an array of solvents. Significant solvatochromic effect on the spectral behavior of the investigated systems was quantified using multiparametric regression analysis along with Density Functional Theory (DFT) calculations. The optical properties of the compounds were seen to be contingent on solvent polarity/polarizability and hydrogen bonding to varying degrees. Assessment of in-vitro anti-acetylcholinesterase (AChE) activity of the investigated systems indicates their efficacy to be even better than FDA approved drug donepezil to treat Alzheimer's disease (AD) and shed novel insights into the biomedical applications of coumarinyl compounds. Interestingly, the AChE inhibition capacity of these compounds showed significant diminution in presence of human serum albumin (HSA), the principal transport protein in blood serum and therefore, reinstated its ability to modulate the therapeutic efficacies of potential drugs. The observed results were validated by quantifying the interaction of the investigated systems with HSA and also investigated through steady state and time-resolved fluorescence spectroscopy along with several other bio-analytical tools. A combination of static and dynamic mechanisms was responsible for the quenching of intrinsic protein fluorescence due to the strong sequestration ability of these coumarin derivatives into the protein binding domain. The observed results emphasized on the need of screening the pharmacological effectiveness of proposed drugs in biologically relevant media rather than in pure buffer.

Promoter of Vegetable Soybean GmTIP1;6 Responds to Diverse Abiotic Stresses and Hormone Signals in Transgenic Arabidopsis.

Tonoplast intrinsic proteins (TIPs), a sub-family of aquaporins (AQPs), are known to play important roles in plant abiotic stress responses. However, evidence for the promoters of TIPs involvement in abiotic stress processes remains scarce. In this study, the promoter of the vegetable soybean GmTIP1;6 gene, which had the highest similarity to TIP1-type AQPs from other plants, was cloned. Expression pattern analyses indicated that the GmTIP1;6 gene was dramatically induced by drought, salt, abscisic acid (ABA), and methyl jasmonate (MeJA) stimuli. Promoter analyses revealed that the GmTIP1;6 promoter contained drought, ABA, and MeJA cis-acting elements. Histochemical staining of the GmTIP1;6 promoter in transgenic Arabidopsis corroborated that it was strongly expressed in the vascular bundles of leaves, stems, and roots. Beta-glucuronidase (GUS) activity assays showed that the activities of the GmTIP1;6 promoter were enhanced by different concentrations of polyethylene glycol 6000 (PEG 6000), NaCl, ABA, and MEJA treatments. Integrating these results revealed that the GmTIP1;6 promoter could be applied for improving the tolerance to abiotic stresses of the transgenic plants by promoting the expression of vegetable soybean AQPs.

Genome-wide identification and biological relevance of broccoli aquaporins.

Broccoli (Brassica oleracea var. italica) is an important crop worldwide, and its regular consumption is associated with health benefits due to the presence of various bioactive compounds. An optimal water balance and homeostasis are needed for plant growth; in this sense, aquaporins play a crucial role. As a result of a genome-wide search, a total of 65 aquaporin genes were identified in broccoli. The aquaporins were classified according to their phylogenetic relationships with other Brassicas species and Arabidopsis thaliana, and evolutionary events of gene duplication were also assessed, highlighting the tendency of NIPs (Nodulin-26-like Intrinsic Proteins) to duplicate. Also, the chromosomal localization, gene duplication, the study of the conserved motifs, and the tertiary structure were determined in broccoli. Functional predictive analyses were also carried out, which, together with the expression analyses in different broccoli plant tissues, allowed the prediction of the biological role of each aquaporin isoform. BoiPIP1-2a and BoiPIP1-2b showed higher expression in all the plant tissues when compared with other aquaporins. BoiTIP1-2b also showed high expression levels and was associated with nitrogen compounds transport such as urea. However, NIPs, through their differential expression and the tandem duplications of the isoforms, were revealed as the putative main actors in the response of broccoli plants to abiotic stress responses. The results of this work pointed to the physiological significance of each aquaporin isoform of broccoli, opening a new field of knowledge and constituting the first step of further in vivo analyses.

The IbPYL8-IbbHLH66-IbbHLH118 complex mediates the abscisic acid-dependent drought response in sweet potato.

Drought limits crop development and yields. bHLH (basic helix-loop-helix) transcription factors play critical roles in regulating the drought response in many plants, but their roles in this process in sweet potato are unknown. Here, we report that two bHLH proteins, IbbHLH118 and IbbHLH66, play opposite roles in the ABA-mediated drought response in sweet potato. ABA treatment repressed IbbHLH118 expression but induced IbbHLH66 expression in the drought-tolerant sweet potato line Xushu55-2. Overexpressing IbbHLH118 reduced drought tolerance, whereas overexpressing IbbHLH66 enhanced drought tolerance, in sweet potato. IbbHLH118 directly binds to the E-boxes in the promoters of ABA-insensitive 5 (IbABI5), ABA-responsive element binding factor 2 (IbABF2) and tonoplast intrinsic protein 1 (IbTIP1) to suppress their transcription. IbbHLH118 forms homodimers with itself or heterodimers with IbbHLH66. Both of the IbbHLHs interact with the ABA receptor IbPYL8. ABA accumulates under drought stress, promoting the formation of the IbPYL8-IbbHLH66-IbbHLH118 complex. This complex interferes with IbbHLH118's repression of ABA-responsive genes, thereby activating ABA responses and enhancing drought tolerance. These findings shed light on the role of the IbPYL8-IbbHLH66-IbbHLH118 complex in the ABA-dependent drought response of sweet potato and identify candidate genes for developing elite crop varieties with enhanced drought tolerance.

Effect of phosphorylation on the structural dynamics, thermal stability of human dopamine transporter: A simulation study using normal modes, molecular dynamics and Markov State Model

The Human Dopamine Transporter (hDAT) plays an essential role in modulating the Influx/Efflux of dopamine, and it is involved in the mechanism of certain neurodegenerative diseases such as Parkinson's disease. Several studies have reported important states for Dopamine transport: outward-facing open state (OFo), the outward-facing closed state (OFc), the holo-occluded state closed (holo), and the inward-facing open state (IFo). Furthermore, experimental assays have shown that different phosphorylation conditions in hDAT can affect the rate of dopamine absorption. We present a protocol using hybrid simulation methods to study the conformational dynamics and stability of states of hDAT under different phosphorylation sites. With this protocol, we explored the conformational space of hDAT, identified the states, and evaluated the free energy differences and the transition probabilities between them in each of the phosphorylation cases. We also presented the conformational changes and correlated them with those described in the literature. There is a thesis/hypothesis that the phosphorylation condition corresponding to NP-333 system (where all sites Ser/Thr from residue 2 to 62 and 254 to 613 are phosphorylated, except residue 333) would decrease the rate of dopamine transport from the extracellular medium to the intracellular medium by hDAT as previously described in the literature by Lin et al., 2003. Our results corroborated this thesis/hypothesis and the data reported. It is probably due to the affectation/changes/alteration of the conformational dynamics of this system that makes the intermediate states more likely and makes it difficult to initial states associated with the uptake of dopamine in the extracellular medium, corroborating the experimental results. Furthermore, our results showed that just single phosphorylation/dephosphorylation could alter intrinsic protein motions affecting the sampling of one or more states necessary for dopamine transport. In this sense, the modification of phosphorylation influences protein movements and conformational preferences, affecting the stability of states and the transition between them and, therefore, the transport.

Transcriptomic analyses of carvone inhibited sprouting in sweet potato (Ipomoea batatas (L.) Lam cv ‘Yan 25′) storage roots

Sprouting is an important postharvest problem in the transport and storage of sweet potato. This study investigated the effect of carvone fumigation on the development of sprouts in ‘Yan 25′ sweet potato storage roots and sprouts. The results showed that visible buds could be observed in untreated root after 4 d at 30 ℃, however no buds were found on carvone treated sweet potato, even after 14 d storage. The levels of β-amylase activity were lower in the carvone treated roots and were associated with higher starch content. Treatment of sweet potato sprouts with carvone severely damaged the sprouts after 24 h and finally turned black after 72 h treatment, resulting in higher MDA content. The transcriptomic analyses showed that the manipulation of auxin distribution, cytokinin metabolism, cell wall synthesis and cell wall loosening processes were associated with carvone inhibition of sprouting in sweet potato roots. Further transcriptomic analyses on sweet potato sprouts revealed a similar transcription profile except that the gibberellin metabolism was also affected in the sprouts by carvone treatment. The up-regulation of apoptosis related genes in carvone treated sprouts, included UDP-D-apiose/UDP-D-xylose synthase (UAXS), ACC oxidase (ACO), tonoplast intrinsic protein (TIP), respiratory burst oxidase (RBOH), senescence associated gene (SAG) which were correlated with the lethal effect of carvone on the sprout. Two conceptual models elucidating the molecular mechanism of carvone inhibited sprouting in sweet potato are proposed. The further elucidation of this mechanism will potentially lead to a theory to optimize the method in sprouting inhibition of sweet potato.

Assessment of root-specific promoters in banana and tobacco and identification of a banana TIP2 promoter with strong root activity.

Genetic modification is one possible strategy to generate bananas (Musa spp.) with resistance to the soil-borne pathogen causing Fusarium wilt. The availability of banana root-specific promoters to target transgene expression to the sites of infection would be beneficial. We have assessed 18 promoter sequences derived from a range of plant species for their expression profiles in banana tissues to identify those with root-specific activity. Promoter sequences were isolated and fused to the β-glucuronidase (GUS) gene to assess their expression levels and tissue specificity in both banana and the model plant tobacco. Two heterologous promoters conferring high root expression levels in banana were identified, including a β‐glucosidase 1 (GLU1) promoter from maize and the RB7-type tonoplast intrinsic protein (TIP)-2 promoter from strawberry. Further, a novel Musa TIP2-2 promoter sequence was isolated and characterized which, when fused to the GUS gene, conferred very high GUS expression levels in banana roots. These promoters will expand the options for the control of gene expression in genetically modified bananas, providing a tool to develop plants with resistance not only to soil-borne diseases such as Fusarium wilt, but also for the improvement of other traits, such as nematode resistance, nutrition or abiotic stress resistance.

Overexpression of the Eucommia~ulmoides Aquaporin, EuPIP1;1, Promotes Leaf Growth, Flowering and Bolting, and Stress Tolerance in Arabidopsis.

Plasma membrane intrinsic protein (PIP) is one of the largest subfamilies of Aquaporins (AQPs) and plays an important role in plant growth and development, and resistance to abiotic stress. In this study, the full length of the EuPIP1;1 cDNA was cloned from Eucommia ulmoides using the rapid amplification of cDNA ends (RACE) method. The EuPIP1;1 gene was induced by drought treatment and expressed in all tested tissues, with the highest expression level in fruit. The subcellular localization showed that EuPIP1;1 was located in the plasma membrane. Constitutive overexpression of EuPIP1;1 in Arabidopsis thaliana could promote leaf growth and development, and accelerate bolting and flowering. Six genes related to growth and flowering (AtPIF4, AtTCP14, AtCRY1, AtCRY2, AtFCA and AtFT) were significantly up-regulated in transgenic lines. Further, EuPIP1;1 gene improved resistance to drought and salt stress in transgenic Arabidopsis. Under drought and salt stress treatment, the transgenic lines had a higher germination rate and accumulation of osmotic substances, lower membrane damage, and could maintain ion homeostasis. Our results suggest that EuPIP1;1 plays an essential role in plant growth and development and in the response to drought and salt stress.

Genome-wide identification of plasma-membrane intrinsic proteins in pumpkin and functional characterization of CmoPIP1-4 under salinity stress

Plasma membrane intrinsic proteins ( PIPs ) play a crucial role in the salinity and drought tolerance of plants. PIPs operate as vital water conduits for regulating water homeostasis during salt stress. Pumpkin is an important Cucurbitaceae crop, and no research on PIPs in pumpkin has been carried out. The current study, presents the first genome-wide characterization of the PIP gene family and functional characterization of CmoPIP1–4 with knockout mutants in pumpkin. According to their phylogeny, 21 CmoPIP genes have been identified and divided into 2 subgroups: CmoPIP1s and CmoPIP2s . Functional studies on their NPA motifs, ar/R filter, and Froger's positions predicted that these CmoPIP proteins would be substrate specific. The CmoPIP1s expression was found to be responsive to salt stress in leaves and roots. CmoPIP1–4 plays an important role in the salt stress tolerance of pumpkin, and the overexpression of CmoPIP1–4 confers salinity tolerance to yeast. However, hyper sensitivity to salt was observed in CmoPIP1–4 CRISPR plants phenotypically (weaker health, lower relative water contents and reduced photosynthetic activities), physiologically (higher Na + /K + ratios, malondialdehyde, ROS and membrane stability index, lower the contents of proline, glycine betaine and also decreased antioxidative enzyme activities), and molecularly (lower expression levels of salt-associated genes CmoSOS1 , CmoHKT1;1 , CmoNHX4 , and a lower expression levels antioxidative genes CmoSOD1/2, CmoPOD1/2 and CmoCAT1/2 ). This study highlights that CmoPIP1–4 is a key player in the stress-signaling pathway in pumpkin plants and could be valuable for developing stress-resistant cucurbit crops in the future. • Twenty-one plasma membrane intrinsic proteins ( PIPs ) were genome-wide characterized in Cucurbita moschata . • Salinity stress decreases the expression of most CmoPIPs . • Overexpression of CmoPIP1-4 conferred salt tolerance in yeast. • A salt-sensitive phenotype was observed after the CmoPIP1-4 knockout in pumpkin roots.

In vitro inhibition of glucose gastro-intestinal enzymes and antioxidant activity of hydrolyzed collagen peptides from different species.

The growing value of industrial collagen by-products has given rise to interest in extracting them from different species of animals. Intrinsic protein structure variation of collagen sources and its hydrolysis can bring about different bioactivities. This study aimed to characterize and evaluate the differences in vitro biological potential of commercial bovine (BH), fish (FH), and porcine hydrolysates (PH) regarding their antioxidant and hypoglycemic activities. All samples showed percentages above 90% of protein content, with high levels of amino acids (glycine, proline, and hydroxyproline), responsible for the specific structure of collagen. The BH sample showed a higher degree of hydrolysis (DH) (8.7%) and a higher percentage of smaller than 2kDa peptides (74.1%). All collagens analyzed in vitro showed inhibition of pancreatic enzymes (α-amylase and α-glucosidase), with the potential to prevent diabetes mellitus. The PH sample showed higher antioxidant activities measured by ORAC (67.08 ± 4.23 μmol Trolox Eq./g) and ABTS radical scavenging (65.69 ± 3.53 μmol Trolox Eq./g) methods. For the first time, DNA protection was analyzed to hydrolyzed collagen peptides, and the FH sample showed a protective antioxidant action to supercoiled DNA both in the presence (39.51%) and in the absence (96.36%) of AAPH (reagent 2,2'-azobis(2-amidinopropane)). The results confirmed that the source of native collagen reflects on the bioactivity of hydrolyzed collagen peptides, probably due to its amino acid composition. PRACTICAL APPLICATIONS: Our data provide new application for collagen hydrolysates with hypoglycemiant and antioxidant activity. These data open discussion for future studies on the additional benefits arising from collagen peptide consumption for the prevention of aging complications or hyperglycemic conditions as observed in chronic diseases such as diabetes mellitus type II (DM 2). The confirmation of these results can open new market areas for the use of collagen with pharmacological applications or to produce new supplements. Furthermore, provides a solution for waste collagen from meat industries and adds value to the product.

In vitro and in vivo anticancer potential and molecular targets of the new colchicine analog IIIM-067

ObjectiveThe current study evaluated various new colchicine analogs for their anticancer activity and to study the primary mechanism of apoptosis and in vivo antitumor activity of the analogs with selective anticancer properties and minimal toxicity to normal cells. MethodsSulforhodamine B (SRB) assay was used to screen various colchicine analogs for their in vitro cytotoxicity. The effect of N-[(7S)-1,2,3-trimethoxy-9-oxo-10-(pyrrolidine-1-yl)5,6,7,9-tetrahydrobenzo[a] heptalene-7-yl] acetamide (IIIM-067) on clonogenicity, apoptotic induction, and invasiveness of A549 cells was determined using a clonogenic assay, scratch assay, and staining with 4′,6-diamidino-2-phenylindole (DAPI) and annexin V/propidium iodide. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels were observed using fluorescence microscopy. Western blot analysis was used to quantify expression of proteins involved in apoptosis, cell cycle, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling. Pharmacokinetic and in vivo efficacy studies against Ehrlich ascites carcinoma (EAC) and Ehrlich solid tumor models were conducted using Swiss albino mice. ResultsIIIM-067 showed potent cytotoxicity and better selectivity than all other colchicine analogs screened in this study. The selective activity of IIIM-067 toward A549 cells was higher among other cancer cell lines, with a selectivity index (SI) value of 2.28. IIIM-067 demonstrated concentration- and time-dependent cytotoxicity against A549 cells with half-maximal inhibitory concentration values of 0.207, 0.150 and 0.106 μmol/L at 24, 48 and 72 h, respectively. It also had reduced toxicity to normal cells (SI > 1) than the parent compound colchicine (SI = 1). IIIM-067 reduced the clonogenic ability of A549 cells in a dose-dependent manner. IIIM-067 enhanced ROS production from 24.6% at 0.05 μmol/L to 82.1% at 0.4 μmol/L and substantially decreased the MMP (100% in control to 5.6% at 0.4 μmol/L). The annexin V-FITC assay demonstrated 78% apoptosis at 0.4 μmol/L. IIIM-067 significantly (P < 0.5) induced the expression of various intrinsic apoptotic pathway proteins, and it differentially regulated the PI3K/AKT/mTOR signaling pathway. Furthermore, IIIM-067 exhibited remarkable in vivo anticancer activity against the murine EAC model, with tumor growth inhibition (TGI) of 67.0% at a dose of 6 mg/kg (i.p.) and a reduced mortality compared to colchicine. IIIM-067 also effectively inhibited the tumor growth in the murine solid tumor model with TGI rates of 48.10%, 55.68% and 44.00% at doses of 5 mg/kg (i.p.), 6 mg/kg (i.p.) and 7 mg/kg (p.o.), respectively. ConclusionIIIM-067 exhibited significant anticancer activity with reduced toxicity both in vitro and in vivo and is a promising anticancer candidate. However, further studies are required in clinical settings to fully understand its potential.

Engineered Protein Nanocages for Concurrent RNA and Protein Packaging In Vivo.

Protein nanocages have emerged as an important engineering platform for biotechnological and biomedical applications. Among naturally occurring protein cages, encapsulin nanocompartments have recently gained prominence due to their favorable physico-chemical properties, ease of shell modification, and highly efficient and selective intrinsic protein packaging capabilities. Here, we expand encapsulin function by designing and characterizing encapsulins for concurrent RNA and protein encapsulation in vivo. Our strategy is based on modifying encapsulin shells with nucleic acid-binding peptides without disrupting the native protein packaging mechanism. We show that our engineered encapsulins reliably self-assemble in vivo, are capable of efficient size-selective in vivo RNA packaging, can simultaneously load multiple functional RNAs, and can be used for concurrent in vivo packaging of RNA and protein. Our engineered encapsulation platform has potential for codelivery of therapeutic RNAs and proteins to elicit synergistic effects and as a modular tool for other biotechnological applications.

The plasma membrane-localized OsNIP1;2 mediates internal aluminum detoxification in rice.

Aluminum (Al) toxicity significantly restricts crop production on acidic soils. Although rice is highly resistant to Al stress, the underlying resistant mechanisms are not fully understood. Here, we characterized the function of OsNIP1;2, a plasma membrane-localized nodulin 26-like intrinsic protein (NIP) in rice. Aluminum stress specifically and quickly induced OsNIP1;2 expression in the root. Functional mutations of OsNIP1;2 in two independent rice lines led to significantly enhanced sensitivity to Al but not other metals. Moreover, the Osnip1;2 mutants had considerably more Al accumulated in the root cell wall but less in the cytosol than the wild-type rice. In addition, compared with the wild-type rice plants, the Osnip1;2 mutants contained more Al in the root but less in the shoot. When expressed in yeast, OsNIP1;2 led to enhanced Al accumulation in the cells and enhanced sensitivity to Al stress, suggesting that OsNIP1;2 facilitated Al uptake in yeast. These results suggest that OsNIP1;2 confers internal Al detoxification via taking out the root cell wall’s Al, sequestering it to the root cell’s vacuole, and re-distributing it to the above-ground tissues.

Biochemical and spectroscopic characterizations of the oligomeric antenna of the coral symbioticSymbiodiniaceae Fugacium kawagutii.

Light-harvesting antennas in photosynthesis capture light energy and transfer it to the reaction centers (RCs) where photochemistry takes place. The sustainable growth of the reef-building corals relies on a constant supply of the photosynthates produced by the endosymbiotic dinoflagellate, belonging to the family of Symbiodiniaceae. The antenna system in this group consists of the water-soluble peridinin-chlorophyll a-protein (PCP) and the intrinsic membrane chlorophyll a-chlorophyll c2-peridinin protein complex (acpPC). In this report, a nonameric acpPC is reported in a dinoflagellate, Fugasium kawagutii (formerly Symbiodinium kawagutii sp. CS-156). We found that extensive biochemical purification altered the oligomerization states of the initially isolated nonameric acpPC. The excitation energy transfer pathways in the acpPC nonamer and its variants were studied using time-resolved fluorescence and time-resolved absorption spectroscopic techniques at 77K. Compared to the well-characterized trimeric acpPC, the nonameric acpPC contains an 11nm red-shifted terminal energy emitter and substantially altered excited state lifetimes of Chl a. The observed energetic overlap of the fluorescence terminal energy emitters with the absorptionof RCs is hypothesized to enable efficient downhill excitation energy transfer. Additionally, the shortened Chl a fluorescence decay lifetime in the oligomeric acpPC indicate a protective self-relaxation strategy. We propose that the highly-oligomerized acpPC nonamer represents an intact functional unit in the Symbiodiniaceae thylakoid membrane. They perform efficient excitation energy transfer (to RCs), and are under manageable regulations in favor of photoprotection.

Whole plant response of Pongamia pinnata to drought stress tolerance revealed by morpho-physiological, biochemical and transcriptome analysis.

Pongamia is considered an important biofuel species worldwide. Drought stress in the early growth stages of Pongamia influences negatively on the germination and seedling development. Due to lack of cultivar stability under drought stress conditions, establishment of successful plantation in drought hit areas becomes a major problem. To address this issue, drought stress response of four Pongamia genotypes was studied at morphological, physio-chemical and transcriptome levels. Drought stress was levied by limiting water for 15 days on three months old seedlings of four genotypes. A significant effect of water stress was observed on the traits considered. The genotype NRCP25 exhibited superior morpho-physiological, biochemical drought responses. Also, the genotype had higher root length, photosynthetic pigments, higher antioxidant enzymes and solute accumulation compared to other genotypes. In addition, transcript profiling of selected drought responsive candidate genes such as trehalose phosphate synthase 1 (TPS1), abscisic acid responsive elements-binding protein 2 (ABF2-2), heat shock protein 17 (HSP 17kDa), tonoplast intrinsic protein 1(TIP 1-2), zinc finger homeodomain protein 2 (ZFP 2), and xyloglucan endotransglucolase 13 (XET 13) showed only up-regulation in NRCP25. Further, the transcriptome responses are in line with key physio-chemical responses exhibited by NRCP25 for drought tolerance. As of now, there are no systematic studies on Pongamia drought stress tolerance; therefore this study offers a comprehensive understanding of whole plant drought stress responsiveness of Pongamia. Moreover, the results support important putative trait indices with potential candidate genes for drought tolerance improvement of Pongamia.

Cannabidivarin alleviates neuroinflammation by targeting TLR4 co-receptor MD2 and improves morphine-mediated analgesia.

Toll-like receptor 4 (TLR4) is a pattern-recognition receptor (PRR) that regulates the activation of immune cells, which is a target for treating inflammation. In this study, Cannabidivarin (CBDV), an active component of Cannabis, was identified as an antagonist of TLR4. In vitro, intrinsic protein fluorescence titrations revealed that CBDV directly bound to TLR4 co-receptor myeloid differentiation protein 2 (MD2). Cellular thermal shift assay (CETSA) showed that CBDV binding decreased MD2 stability, which is consistent with in silico simulations that CBDV binding increased the flexibility of the internal loop of MD2. Moreover, CBDV was found to restrain LPS-induced activation of TLR4 signaling axes of NF-κB and MAPKs, therefore blocking LPS-induced pro-inflammatory factors NO, IL-1β, IL-6 and TNF-α. Hot plate test showed that CBDV potentiated morphine-induced antinociception. Furthermore, CBDV attenuated morphine analgesic tolerance as measured by the formalin test by specifically inhibiting chronic morphine-induced glial activation and pro-inflammatory factors expression in the nucleus accumbent. This study confirms that MD2 is a direct binding target of CBDV for the anti-neuroinflammatory effect and implies that CBDV has great translational potential in pain management.

Conserved conformational dynamics determine enzyme activity.

Homologous enzymes often exhibit different catalytic rates despite a fully conserved active site. The canonical view is that an enzyme sequence defines its structure and function and, more recently, that intrinsic protein dynamics at different time scales enable and/or promote catalytic activity. Here, we show that, using the protein tyrosine phosphatase PTP1B, residues surrounding the PTP1B active site promote dynamically coordinated chemistry necessary for PTP1B function. However, residues distant to the active site also undergo distinct intermediate time scale dynamics and these dynamics are correlated with its catalytic activity and thus allow for different catalytic rates in this enzyme family. We identify these previously undetected motions using coevolutionary coupling analysis and nuclear magnetic resonance spectroscopy. Our findings strongly indicate that conserved dynamics drives the enzymatic activity of the PTP family. Characterization of these conserved dynamics allows for the identification of novel regulatory elements (therapeutic binding pockets) that can be leveraged for the control of enzymes.