Direct measure of functional importance visualized atom-by-atom for photoactive yellow protein: application to photoisomerization reaction

Photoreceptor proteins serve as efficient nano-machines for the photoenergy conversion and the photosignal transduction of living organisms. For instance, the photoactive yellow protein derived from a halophilic bacterium has the p-coumaric acid chromophore, which undergoes an ultrafast photoisomerization reaction after light illumination. To understand the structure-function relationship at the atomic level, we used a computational method to find functionally important atoms for the photoisomerization reaction of the photoactive yellow protein. In the present study, a "direct" measure of the functional significance was quantitatively evaluated for each atom by calculating the partial atomic driving force for the photoisomerization reaction. As a result, we revealed the reaction mechanism in which the specific role of each functionally important atom has been well characterized in a systematic manner. In addition, we observed that this mechanism is strongly conserved during the thermal fluctuation of the photoactive yellow protein. We compared the experimental data of fluorescence decay constant of several different mutants and the present analysis. As a result, we found that the reaction rate constant is decreased when a large positive driving force is missing.     

A desolvation barrier to hydrophobic cluster formation may contribute to the rate-limiting step in protein folding.

To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error.     

Signal transduction in the photoactive yellow protein. I. Photon absorption and the isomerization of the chromophore

Molecular dynamics simulation techniques together with time-dependent density functional theory calculations have been used to investigate the effect of photon absorption by a 4-hydroxy-cinnamic acid chromophore on the structural properties of the photoactive yellow protein (PYP) from Ectothiorodospira halophila. The calculations suggest that the protein not only modifies the absorption spectrum of the chromophore but also regulates the subsequent isomerization of the chromophore by stabilizing the isomerization transition state. Although signaling from PYP is thought to involve partial unfolding of the protein, the mechanical effects accompanying isomerization do not appear to directly destabilize the protein.     

Identification and characterization of protein subcomplexes in yeast

Protein complexes are major components of cellular organization. Based on large-scale protein complex data, we present the first statistical procedure to find insightful substructures in protein complexes: we identify protein subcomplexes (SCs), i.e., multiprotein assemblies residing in different protein complexes. Four protein complex datasets with different origins and variable reliability are separately analyzed. Our method identifies well-characterized protein assemblies with known functions, thereby confirming the utility of the procedure. In addition, we also identify hitherto unknown functional entities consisting of either functionally unknown proteins or proteins with different functional annotation. We show that SCs represent more reliable protein assemblies than the original complexes. Finally, we demonstrate unique properties of subcomplex proteins that underline the distinct roles of SCs: (i) SCs are functionally and spatially more homogeneous than complete protein complexes (this fact is utilized to predict functional roles and subcellular localizations for so far unannotated proteins); (ii) the abundance of subcomplex proteins is less variable than the abundance of other proteins; (iii) SCs are enriched with essential and synthetic lethal proteins; and (iv) mutations in SC-proteins have higher fitness effects than mutations in other proteins.     

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila,with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.     

Monitoring the interference of protein-protein interactions in vivo by bimolecular fluorescence complementation: the DnaK case

Many cellular processes depend on protein-protein interactions. The identification of molecules able to modulate protein contacts is of significant interest for drug discovery and chemical biology. Nevertheless, finding antagonists of protein interactions that work efficiently within the cell is a challenging task. Here, we describe the novel use of bimolecular fluorescence complementation (BIFC) to detect compounds that block the interaction of target proteins in vivo. In the BIFC method, each interaction partner is fused to a complementary fragment of a fluorescent protein and interactions are detected by fluorescence restoration after reporter reassembly. Here, we demonstrate that the inhibition of specific intracellular protein interactions results in a concomitant decrease in fluorescence emission. We also show that integration of BIFC with flow cytometry might provide an effective means to detect interaction modulators by directly reading out changes in the reporter signal. The in vivo application of this approach is illustrated through monitoring the inhibition of the interaction between the Escherichia coli Hsp70 chaperone and a short peptidic substrate by pyrrhocoricin-derived antibacterial peptides.     

Role of the 14-3-3 C-terminal loop in ligand interaction

14-3-3 proteins are a family of conserved dimeric molecules that interact with a broad range of target proteins, most of which contain phosphoserine/threonine. The amphipathic groove of 14-3-3 is the main structural feature involved in mediating its associations. We have studied another domain of 14-3-3, the C-terminal loop, to determine what role it plays in ligand interaction. A truncated form of 14-3-3zeta lacking this C-terminal loop was generated and found to bind with higher affinity than the wild-type 14-3-3zeta protein to the ligands Raf-1 and Bad. Interestingly, the truncated 14-3-3zeta also showed increased association with the 14-3-3 binding-deficient Bad/S136A mutant. Taken together, these data support a role for the C-terminal loop as a general inhibitor of 14-3-3/ligand interactions. This may provide a mechanism by which inappropriate associations with 14-3-3 are prevented.     

Role of calcineurin and protein phosphatase-2A in the regulation of DARPP-32 dephosphorylation in neostriatal neurons

DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cyclic AMP-dependent protein kinase, resulting in its conversion to a potent inhibitor of protein phosphatase-1 (PP-1). Conversely, Thr34-phosphorylated DARPP-32 is dephosphorylated and inactivated in vitro by calcineurin and protein phosphatase-2A (PP-2A). We have investigated the relative contributions of these protein phosphatases to the regulation of DARPP-32 dephosphorylation in mouse neostriatal slices. Cyclosporin A (5 microM), a calcineurin inhibitor, maximally increased the level of phosphorylated DARPP-32 by 17+/-2-fold. Okadaic acid (1 microM), an inhibitor of PP-1 and PP-2A, had a smaller effect, increasing phospho-DARPP-32 by 5.1+/-1.3-fold. The effect of okadaic acid on DARPP-32 phosphorylation was shown to be due to inhibition of PP-2A activity. Incubation of slices in the presence of cyclosporin A plus either okadaic acid or calyculin A, another PP-1/PP-2A inhibitor, caused a synergistic increase in the level of phosphorylated DARPP-32. The use of Ca2(+)-free/EGTA medium mimicked the effects of cyclosporin A on DARPP-32 phosphorylation, supporting the conclusion that the action of cyclosporin on DARPP-32 phosphorylation was attributable to blockade of the Ca2(+)-dependent activation of calcineurin. The results indicate that calcineurin and PP-2A, but not PP-1, act synergistically to maintain a low level of phosphorylated DARPP-32 in neostriatal slices.     

S-layer protein transport across the cell wall of Corynebacterium glutamicum: in vivo kinetics and energy requirements

Corynebacteria are Gram-positive bacteria with a very peculiar cell envelope structure as it is constituted of an inner membrane and an outer membrane-like structure. Protein secretion in Corynebacterium glutamicum was studied in vivo, using the S-layer protein PS2 as a model. We show that different variants of PS2 protein are exported through the whole cell envelope with a half-life ranging between 2 and 4 min, by a two-step mechanism. The first step, which is over after about 1.5 min, is ATP- and proton motive force-dependent and may correspond to translocation across the inner membrane via the 'Sec' machinery. The second step, across the cell wall and the outer mycolate layer, is rapid but independent of energy sources. This very efficient secretion process across the mycolate layer raises the question of the existence in this layer of a specific machinery.     

Role of phenolic acids and enzymes of phenylpropanoid pathway in resistance of chayote fruit (Sechium edule) against infestation by melon fly,Bactrocera cucurbitae

Melon fly is a serious pest of cucurbits all over the world causing huge losses to yield. However, the only exception is the chayote fruit (Sechium edule) that shows resistance to melon fly infestation. Studies on culture of melon fly indicated the absence of plant traits resisting oviposition on chayote fruit. However, the melon fly was unable to complete its life cycle successfully on chayote showing that factors inhibiting larval development in melon fly could be attributed to biochemical constituents. Studies were, therefore, carried out to compare the biochemical responses of chayote, a melon fly resistant species and bitter gourd, a susceptible species to melon fly infestation with regard to the levels of phenolic acids and activities of the enzymes of phenylpropanoid pathway (PPP) leading to synthesis of lignin. The resistant chayote exhibited significantly higher accumulation of lignin associated with higher activities of phenylalanine ammonia‐lyase (PAL), tyrosine ammonia‐lyase (TAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD). On the contrary, the susceptible bitter gourd recorded lower activities of PAL, CAD and POD and a decreasing trend of TAL during infestation associated with a lower lignin content. The monomer composition of lignin in the resistant chayote showed twofold higher level of guaiacyl (G) and syringyl (S) units compared to susceptible bitter gourd and the G/S ratio during infestation increased in chayote while decreasing in bitter gourd. The levels of PPP intermediates, p‐coumaric acid was higher in chayote while p‐hydroxy benzoic acid, a chemo‐attractant, was higher in bitter gourd. Incorporation of p‐coumaric acid in the larval diet strongly inhibited larval growth even as p‐hydroxy benzoic acid promoted growth confirming the direct role of p‐coumaric acid in conferring resistance to chayote. The level of salicylic acid, a signal molecule involved in induction of defence response, was higher in chayote compared to bitter gourd. Chayote also exhibited higher level of activity of POD in the phloem exudates compared to bitter gourd. The higher concentration of sugars in exudates of chayote might act like signalling molecules causing activation of plant genes, especially of the phenylpropanoid biosynthesis pathway and possibly produce an osmotic effect inducing resistance against the melon fly. Thus, the study revealed that the resistance in chayote to melon fly infestation is a complex, multi‐layered process in which the activities of PPP enzymes generating phenolic intermediates leading to lignin biosynthesis and the composition of exudates appear to play significant roles. Besides, the study also indicated that different forms of lignin might play a role in the resistance of chayote against melon fly infestation.     

Role of local germplasm and induced mutations in the improvement of the protein content in rice

Summary Ninety local cultivars and 124 induced grain shape mutants were screened for their protein content and the distribution of protein in their endosperm. The protein content varied widely from 4.2 to 12.1% in the local varieties and from 5.5 to 13.7% in the mutants. Some high protein lines like Chinnavadlu, Budumavadlu, etc. with more than 10.0% protein were identified. Protein distribution studies have shown that several mutants, such as IR-8-FG-28, IR-8-FG-33, etc., have a deeper distribution of protein bodies and some local cultivars, like Kakathya and Muchulu, have a relatively more uniform distribution of protein in the endosperm. The protein content at different milling levels revealed that at 12% milling the percentage retention of protein varied from about 79 to 96% irrespective of the percentage of protein, suggesting that the distribution of protein, in addition to the protein content, plays an important role in nutritive value of rice.     

Systematic localisation of proteins fused to the green fluorescent protein in Bacillus subtilis: identification of new proteins at the DNA replication factory

Construction and microscopic imaging of protein fusions to green fluorescent protein (GFP) have revolutionised our understanding of bacterial structure and function. We have undertaken a systematic study of the localisation of over 100 Bacillus subtilis proteins, following the development of high-throughput construction and analysis procedures. We focused on proteins linked in various ways to the DNA replication machinery, as well as on proteins exemplifying a range of other cellular functions and structures. The results validate the approach as a way of obtaining systematic protein localisation information. They also provide a range of novel biological insights, particularly through the identification of a number of proteins not previously known to be associated with the DNA replication factory.     

Role of p38 protein kinase in the ligand-independent ubiquitination and down-regulation of the IFNAR1 chain of type I interferon receptor

Phosphorylation-dependent ubiquitination and degradation of the IFNAR1 chain of type I interferon (IFN) receptor is a robust and specific mechanism that limits the magnitude and duration of IFNα/β signaling. Besides the ligand-inducible IFNAR1 degradation, the existence of an "inside-out" signaling that accelerates IFNAR1 turnover in the cells undergoing the endoplasmic reticulum (ER) stress and activated unfolded protein responses has been recently described. The latter pathway does not require either presence of ligands (IFNα/β) or catalytic activity of Janus kinases (JAK). Instead, this pathway relies on activation of the PKR-like ER kinase (PERK) and ensuing specific priming phosphorylation of IFNAR1. Here, we describe studies that identify the stress activated p38 protein kinase as an important regulator of IFNAR1 that acts downstream of PERK. Results of the experiments using pharmacologic p38 kinase inhibitors, RNA interference approach, and cells from p38α knock-out mice suggest that p38 kinase activity is required for priming phosphorylation of IFNAR1 in cells undergoing unfolded protein response. We further demonstrate an important role of p38 kinase in the ligand-independent stimulation of IFNAR1 ubiquitination and degradation and ensuing attenuation of IFNα/β signaling and anti-viral defenses. We discuss the distinct importance of p38 kinase in regulating the overall responses to type I IFN in cells that have been already exposed to IFNα/β versus those cells that are yet to encounter these cytokines.     

Role of phospholipase D in the activation of protein kinase D by lysophosphatidic acid

Protein kinase D was auto-phosphorylated at Ser916 and trans-phosphorylated at Ser744/Ser748 in Rat-2 fibroblasts treated with lysophosphatidic acid. Both phosphorylations were inhibited by 1-butanol, which blocks phosphatidic acid formation by phospholipase D. The phosphorylations were also reduced in Rat-2 clones with decreased phospholipase D activity. Platelet-derived growth factor-induced protein kinase D phosphorylation showed a similar requirement for phospholipase D, but that induced by 4beta-phorbol 12 myristate 13-acetate did not. Propranolol an inhibitor of diacylglycerol formation from phosphatidic acid blocked the phosphorylation of protein kinase D, whereas dioctanoylglycerol induced it. The temporal pattern of auto-phosphorylation of protein kinase D closely resembled that of phospholipase D activation and preceded the trans-phosphorylation by protein kinase C. These results suggest that protein kinase D is activated by lysophosphatidic acid through sequential phosphorylation and that diacylglycerol produced by PLD via phosphatidic acid is required for the autophosphorylation that occurs prior to protein kinase C-mediated phosphorylation.     

Role of the F-box protein Skp2 in adhesion-dependent cell cycle progression.

Cell adhesion to the extracellular matrix (ECM) is a requirement for proliferation that is typically lost in malignant cells. In the absence of adhesion, nontransformed cells arrest in G1 with increased levels of the cyclin-dependent kinase inhibitor p27. We have reported previously that the degradation of p27 requires its phosphorylation on Thr-187 and is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCF(Skp2) ubiquitin ligase complex. Here, we show that the accumulation of Skp2 protein is dependent on both cell adhesion and growth factors but that the induction of Skp2 mRNA is exclusively dependent on cell adhesion to the ECM. Conversely, the expression of the other three subunits of the SCF(Skp2) complex is independent of cell anchorage. Phosphorylation of p27 on Thr-187 is also not affected significantly by the loss of cell adhesion, demonstrating that increased p27 stability is not dependent on p27 dephosphorylation. Significantly, ectopic expression of Skp2 in nonadherent G1 cells resulted in p27 downregulation, entry into S phase, and cell division. The ability to induce adhesion-independent cell cycle progression was potentiated by coexpressing Skp2 with cyclin D1 but not with cyclin E, indicating that Skp2 and cyclin D1 cooperate to rescue proliferation in suspension cells. Our study shows that Skp2 is a key target of ECM signaling that controls cell proliferation.     

Role of the yeast DNA repair protein Nej1 in end processing during the repair of DNA double strand breaks by non-homologous end joining

DNA double strand breaks (DSB)s often require end processing prior to joining during their repair by non-homologous end joining (NHEJ). Although the yeast proteins, Pol4, a Pol X family DNA polymerase, and Rad27, a nuclease, participate in the end processing reactions of NHEJ, the mechanisms underlying the recruitment of these factors to DSBs are not known. Here we demonstrate that Nej1, a NHEJ factor that interacts with and modulates the activity of the NHEJ DNA ligase complex (Dnl4/Lif1), physically and functionally interacts with both Pol4 and Rad27. Notably, Nej1 and Dnl4/Lif1, which also interacts with both Pol4 and Rad27, independently recruit the end processing factors to in vivo DSBs via mechanisms that are additive rather than redundant. As was observed with Dnl4/Lif1, the activities of both Pol4 and Rad27 were enhanced by the interaction with Nej1. Furthermore, Nej1 increased the joining of incompatible DNA ends in reconstituted reactions containing Pol4, Rad27 and Dnl4/Lif1, indicating that the stimulatory activities of Nej1 and Dnl4/Lif1 are also additive. Together our results reveal novel roles for Nej1 in the recruitment of Pol4 and Rad27 to in vivo DSBs and the coordination of the end processing and ligation reactions of NHEJ.     

Reality of P-680 chlorophyll protein – Identification of the site of primary photochemistry in oxygenic photosynthesis

Until recently nearly all available experimental evidence seemed to indicate that the largest subunit of about 50 kDa in the photosystem II core complex ( psb B gene product) is the site of primary photochemistry, and thus the name "P-680 apoprotcin" has been given to this subunit. The notion, however, has also been challenged on the basis of deduced amino acid sequence homology between the proteins in the photosystem II and those of the purple bacterial reaction center. The actual preparation of a functionally active photosystem II reaction center completely devoid of the psb B gene product, but consisting of D-1 and D-2 proteins and cytochrome b -559, has now been achieved.     

Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.