Piotr Dobryszycki

Influence of silybin on biophysical properties of phospholipid bilayers

AbstractAim:Silybin (silibinin) is major biologically active flavonolignan extracted from milk thistle (Sylibum marianum). Its biological activities include hepato-protection, anticancer properties, and antioxidant- and membrane-stabilizing functions. Although membranes are postulated to be one of the cellular targets for silybin, little is known about its interaction with phospholipid bilayers.Methods:In the present work, the interactions of silybin with phosphatidylcholine bilayers were studied in detail using fluorescence spectroscopy, microcalorimetry and electron spin resonance techniques.Results:The results showed that silybin interacted with the surface of lipid bilayers. It affected the generalized polarization of the fluorescent probe Prodan, while not influencing the more deeply located Laurdan. Silybin lowered the main phospholipid phase transition temperature as judged by microcalorimetry, and caused the immobilization of spin probe Tempo-palmitate located on the surface of membranes. The mobility of spin probes 5- and 16-doxyl stearic acid was not affected by silybin. Silybin-induced quenching of 1,6-diphenyl-1,3,5-hexatriene fluorescence indicated that some flavonoid molecules partitioned into the hydrophobic region of membranes, which did not change significantly the biophysical properties of the deeper membrane regions.Conclusion:Such a behavior of silybin in membranes is in accordance with its postulated biological functions and neglectable side effects of therapies using silybin.

Dual FRET assay for detecting receptor protein interaction with DNA

We present here a new assay that is based on the idea of the molecular beacon. This assay makes it possible to investigate two proteins interacting with DNA at two binding sites that are close to each other. The effectiveness of the test depends on the exclusive binding of three DNA fragments in the presence of two proteins, and the monitoring of the process depends upon observing the quenching of two independent fluorescence donors. As a model we used the components of the heterodimeric ecdysteroid receptor proteins ultraspiracle (Usp) and ecdysone receptor (EcR) from Drosophila melanogaster and a response element from the promoter of the hsp27 gene. The response element consists of two binding sites (half-sites) for the DNA binding domains (DBDs). We have shown that protein–protein interactions mediate cooperative binding of the ecdysteroid receptor DBDs to a hsp27pal response element. The analysis of the microscopic dissociation constants obtained with the DMB led to the conclusion that there was increased affinity of UspDBD to the 5′ half-site in the presence of EcRDBD when the 3′ half-site was occupied, and increased affinity of EcRDBD to the 3′ half-site when the 5′ half-site was occupied.

Crystallization and preliminary crystallographic studies of juvenile hormone-binding protein from Galleria mellonella haemolymph

Juvenile hormone-binding protein (JHBP) isolated from Galleria mellonella haemolymph has been crystallized using the hanging-drop method in two polymorphic forms. The best diffracting crystals (2.7 A) are trigonal, space group P3(1)21 (or P3(2)21), with unit-cell parameters a = 110.4, c = 93.9 A. X-ray diffraction data have been collected for the native crystals using synchrotron radiation and cryogenic conditions (100 K).

The high-adhesive properties of the FimH adhesin of Salmonella enterica serovar Enteritidis are determined by a single F118S substitution

The binding properties of low- and high-adhesive forms of FimH adhesins from Salmonella enterica serovars Enteritidis and Typhimurium (S. Enteritidis and S. Typhimurium) were studied using chimeric proteins containing an additional peptide that represents an N-terminal extension of the FimF protein. This modification, by taking advantage of a donor strand exchange mechanism, closes the hydrophobic groove in the fimbrial domain of the FimH adhesin. Such self-complemented adhesins (scFimH) did not form aggregates and were more stable (resistant to proteolytic cleavage) than native FimH. High-adhesive variants of scFimH proteins, with alanine at position 61 and serine at position 118, were obtained by site-directed mutagenesis of fimH genes from low-adhesive variants of S. Enteritidis and S. Typhimurium, with glycine at position 61 and phenylalanine at position 118. Direct kinetic analysis using surface plasmon resonance (SPR) and glycoproteins carrying high-mannose carbohydrate chains (RNase B, horseradish peroxidase and mannan-BSA) revealed the existence of high- and low-adhesive allelic variants, not only in S. Typhimurium but also in S. Enteritidis. Using two additional mutants of low-adhesive FimH protein from S. Enteritidis (Gly61Ala and Phe118Ser), SPR analysis pointed to Ser118 as the major determinant of the high-adhesive phenotype of type 1 fimbriae from S. Enteritidis. These studies demonstrated for the first time that the functional differences observed with whole fimbriated bacteria could be reproduced at the level of purified adhesin. They strongly suggest that the adhesive properties of type 1 fimbriae are determined only by structural differences in the FimH proteins and are not influenced by the fimbrial shaft on which the adhesin is located.

Intrinsic disorder of Drosophila melanogaster hormone receptor 38 N-terminal domain

Drosophila hormone receptor 38 (dHR38), an ortholog of the vertebrate NR4A subclass of nuclear receptors, responds to ecdysteroids, which mediate developmental transitions during the Drosophila life cycle. However, this response is independent of the ecdysteroid receptor, and it does not involve binding of ecdysteroids to dHR38. It has been suggested that ecdysteroids may indirectly activate dHR38, perhaps by recruiting specific proteins. There have been recent reports pointing out the decisive role that nuclear receptor N‐terminal domains (NTDs) have in protein–protein interactions that are important for regulation of gene expression. It is reasonable to assume that dHR38‐NTD may also be involved in some protein–protein interactions that are critical for the ecdysteroid signaling pathway. To facilitate the exploration of the molecular basis of these interactions, we developed and optimized a protocol for the efficient expression and purification of the recombinant dHR38‐NTD. Using a diverse array of biochemical and biophysical methods, we carried out the first structural characterization of dHR38‐NTD. The results of our study indicate that dHR38‐NTD exhibits a characteristic reminiscent of pre‐molten globule‐like intrinsically disordered proteins existing in a partially unfolded conformation with regions of secondary structures. The dHR38‐NTD structure, which apparently comprises some local, ordered, tertiary structure clusters, is pliable and can adopt more ordered conformations in response to changes in environmental conditions. Thus, dHR38‐NTD, which exhibits the structural and functional characteristic of a pre‐molten globule‐like intrinsically disordered protein, could serve as a platform for multiple protein–protein interactions, possibly including interactions with proteins involved in an unusual ecdysteroid signaling pathway. Proteins 2011.© 2010 Wiley‐Liss, Inc.

Calcium Ion Binding Properties and the Effect of Phosphorylation on the Intrinsically Disordered Starmaker Protein

Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stms affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 μM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 μM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

Effect of acrylamide on aldolase structure. II. Characterization of aldolase unfolding intermediates.

Molecules of muscle aldolase A exposed to acrylamide change their conformation via I1, T, I2, D intermediates [1] and undergo a slow irreversible chemical modification of thiol groups. There is no direct correlation between activity loss and thiol groups modification. In the native enzyme two classes of Trp residues of 1. 8 ns and 4.9 ns fluorescence lifetime have been found. Acrylamide (0. 2-0.5 M) increases lifetime of longer-lived component, yet the transfer of aldolase molecules even from higher (1.0 M) perturbant concentration to a buffer, allows regain original Trp fluorescence lifetime. I1, detected at about 0.2 M acrylamide, represents low populated tetramers of preserved enzyme activity. T, of maximum population at about 0.7-1.0 M acrylamide, consists of meta-stable tetramers of partial enzymatic activity. These molecules are able to exchange their subunits with aldolase C in opposition to the native molecules. At transition point for I2 appearance (1.8 M acrylamide), aldolase becomes highly unstable: part of molecules dissociate into subunits which in the absence of perturbant are able to reassociate into active tetramers, the remaining part undergoes irreversible denaturation and aggregation. Some expansion of aldolase tetramers takes place prior to dissociation. D, observed above 3.0 M acrylamide, consists of irreversibly denatured enzyme molecules.

The Application of an Immobilized Molecular Beacon for the Analysis of the DNA Binding Domains from the Ecdysteroid Receptor Proteins Usp and EcR's Interaction with the hsp27 Response Element

The nonstandard molecular beacon described in this article consists of 2 fragments, each built of a short single-stranded oligonucleotide sequence and a double-stranded sequence. One of these hybridization probes, labeled with a fluorescence donor (fluorescein), is solid phase immobilized. The second nonimmobilized probe is labeled with a fluorescence quencher (dabcyl). Annealing of both probes via single-stranded sequences was possible only in the presence of a specific protein molecule that recognized the response element sequence initially separated between the immobilized and nonimmobilized fragments. The system was applied successfully to detect the sequence-specific interaction of a natural hsp27 response element from the promoter of the hsp27 gene with the DNA binding domains of 2 nuclear receptor proteins: ultraspiracle Usp (UspDBD) and the ecdysone receptor EcR (EcRDBD). Measured in the absence of EcRDBD, the dissociation constant, Kd of the UspDBD-hsp27 complex, was determined to be 3.26 nM, whereas for UspDBD devoid of the A-box (UspDBDΔAhsp27 ), the dissociation constant was 4.81 nM. The respective Kd values in the presence of EcRDBD were 2.43 nM and 10.80 nM. The results obtained with the immobilized molecular beacon technology were in agreement with those obtained by conventional fluorescence titrations and by fluorescence resonance energy transfer measurements with nonimmobilized beacons. ( Journal of Biomolecular Screening 2008:899-905)

Effect of acrylamide on aldolase structure. I. Induction of intermediate states.

Acrylamide is a fluorescence quencher frequently applied for analysis of protein fluorophores exposure with the silent assumption that it does not affect the native structure of protein. In this report, it is shown that quenching of tryptophan residues in aldolase is a time-dependent process. The Stern-Volmer constant increases from 1.32 to 2.01 M-1 during the first 100 s of incubation of aldolase with acrylamide. Two tryptophan residues/subunit are accessible to quenching after 100 s of aldolase interaction with acrylamide. Up to about 1.2 M acrylamide concentration enzyme inactivation is reversible. Independent analyses of the changes of enzyme activity, 1ANS fluorescence during its displacement from aldolase active-site, UV-difference spectra and near-UV CD spectra were carried out to monitor the transition of aldolase structure. From these measurements a stepwise transformation of aldolase molecules from native state (N) through intermediates: I1, T, I2, to denatured (D) state is concluded. The maxima of I1, T, I2 and D states populations occur at 0.2, 1.0, 2.0 and above 3.0 M of acrylamide concentration, respectively. Above 3.5 M, acrylamide aldolase molecules become irreversibly inactivated.

The composite nature of the interaction between nuclear receptors EcR and DHR38

Abstract Ecdysteroids coordinate essential biological processes in Drosophila through a complex of two nuclear receptors, the ecdysteroid receptor (EcR) and the ultraspiracle protein (Usp). Biochemical experiments have shown that, in contrast to Usp, the EcR molecule is characterized by high intramolecular plasticity. To investigate whether this plasticity is sufficient to form EcR complexes with nuclear receptors other than Usp, we studied the interaction of EcR with the DHR38 nuclear receptor. Previous in vitro experiments suggested that DHR38 can form complexes with Usp and thus disrupt Usp-EcR interaction with the specific hsp27pal response element. This article provides the experimental evidence that EcR is able to form complexes with DHR38 as well. The recombinant DNA-binding domains (DBDs) of EcR and DHR38 interact specifically on hsp27pal. However, the interaction between the receptors is not restricted to their isolated DBDs. We pre\xadsent data that indicate that the full-length EcR and DHR38 can also form specific complexes within the nuclei of living cells. This interaction is mediated by the hinge region of EcR, which was recently classified as an intrinsically disordered region. Our results indicate that DHR38 might modulate the activity of the Usp-EcR heterodimer by forming complexes with both of its components.