Agnieszka Polit

Hetero-dimerization of serotonin 5-HT2A and dopamine D2 receptors

In the present study, detailed information is presented on the hetero-dimerization of the serotonin 5-HT(2A) receptor and the dopamine D(2) receptor. Biophysical approaches (fluorescence spectroscopy as well as fluorescence lifetime microscopy) were used to determine the degree of fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent protein labeled receptor variants co-expressed in human embryonic kidney 293 cells (HEK293). Recorded data demonstrate the existence of energy transfer between the wild-type forms of 5-HT(2A)R and D(2)R, pointing toward the formation of hetero-5-HT(2A)R/D(2)R dimers and homo-5-HT(2A)R/5-HT(2A)R dimers. Moreover, the present study investigates the role of specific motifs (one containing adjacent arginine residues (217RRRRKR222) in the third intracellular loop (ic3) of D(2)R, and the other consisting of acidic glutamate residues (454EE455) in the C-tail of (5-HT(2A)R) in the formation of noncovalent complexes between these receptors. Our results suggest that these regions of 5-HT(2A)R and D(2)R may be involved in the interaction between these two proteins. On the other hand, the above-mentioned motifs do not play an important role in the homo-dimerization of these receptors. Furthermore, we estimated the influence of specific receptor ligands on the dimerization processes. Agonists (DOI and quinpirole) and antagonists (ketanserin and butaclamol) cause different effects on FRET efficiency depending on whether homo- or hetero-complexes are present. These data may have therapeutic implications, since (using the immunofluorescence double labeling protocols) the co-localization of these two receptors was demonstrated in the medial prefrontal cortex and pars reticulate of the substantia nigra of the rat brain.

Two modes of fatty acid binding to bovine β‐lactoglobulin—crystallographic and spectroscopic studies

Lactoglobulin is a natural protein present in bovine milk and common component of human diet, known for binding with high affinity wide range of hydrophobic compounds, among them fatty acids 12–20 carbon atoms long. Shorter fatty acids were reported as not binding to β‐lactoglobulin. We used X‐ray crystallography and fluorescence spectroscopy to show that lactoglobulin binds also 8‐ and 10‐carbon caprylic and capric acids, however with lower affinity. The determined apparent association constant for lactoglobulin complex with caprylic acid is 10.8 ± 1.7 × 103 M−1, while for capric acid is 6.0 ± 0.5 × 103 M−1. In crystal structures determined with resolution 1.9 Å the caprylic acid is bound in upper part of central calyx near polar residues located at CD loop, while the capric acid is buried deeper in the calyx bottom and does not interact with polar residues at CD loop. In both structures, water molecule hydrogen‐bonded to carboxyl group of fatty acid is observed. Different location of ligands in the binding site indicates that competition between polar and hydrophobic interactions is an important factor determining position of the ligand in β‐barrel. Copyright

Structural and thermodynamic studies of binding saturated fatty acids to bovine β-lactoglobulin ☆

Lactoglobulin is a globular milk protein for which physiological function has not been clarified. Due to its binding properties lactoglobulin might serve as a carrier for bioactive molecules. Binding of 12-, 14-, 16- and 18-carbon saturated fatty acids to bovine β-lactoglobulin has been characterised by isothermal titration calorimetry and X-ray crystallography as a part of systematic studies of lactoglobulin complexes with ligands of biological importance. The thermodynamic parameters have been determined for lauric, myristic and palmitic acid complexes revealing systematic decrease of enthalpic and increase of entropic component of ΔG with elongation of aliphatic chain. In all crystal structures determined with resolution 1.9-2.1Å, single fatty acid molecule was found in the β-barrel in extended conformation with individual pattern of interactions. Location of a fatty acid in the binding site depends on the length of aliphatic chain and influences polar interactions between protein and ligand. Systematic changes of entropic component indicate important role of water in binding process.

cAMP Receptor Protein from Escherichia coli as a Model of Signal Transduction in Proteins – A Review

In Escherichia coli, cyclic AMP receptor protein (CRP) is known to regulate the transcription of about 100 genes. The signal to activate CRP is the binding of cyclic AMP. It has been suggested that binding of cAMP to CRP leads to a long-distance signal transduction from the N-terminal cAMP-binding domain to the C-terminal domain of the protein, which is responsible for interaction with specific sequences of DNA. The signal transduction plays a crucial role in the activation of the protein. The most sophisticated spectroscopic techniques, other techniques frequently used in structural biochemistry, and site-directed mutagenesis have been used to investigate the details of cAMP-mediated allosteric control over CRP conformation and activity as a transcription factor. The aim of this review is to summarize recent works and developments pertaining to cAMP-dependent CRP signal transduction in E. coli.

Binding of 18-carbon unsaturated fatty acids to bovine β-lactoglobulin—Structural and thermodynamic studies

Binding of 18-carbon unsaturated oleic and linoleic acid to lactoglobulin, the milk protein, has been studied for the first time by isothermal titration calorimetry (ITC) and X-ray crystallography. Crystal structures determined to resolution 2.10 Å have revealed presence of single fatty acid molecule bound in β-barrel, the primary binding site, with carboxyl group hydrogen bonded to Glu62. The aliphatic chain of both ligands is in almost linear conformation and their interactions with the protein are similar to observed in structure of lactoglobulin with stearic acid. The ITC experiments showed that binding of unsaturated fatty acids to LGB is spontaneous and exothermic. The stoichiometry of binding is lower than 1.0, association constant is 9.7 × 10(5)M(-1) and 9.0 × 10(5)M(-1) for oleic and linoleic acid, respectively. Solvent relief seems to be the major contributor to entropic changes upon fatty acid binding to lactoglobulin.

Conformational variability of goat β-lactoglobulin: crystallographic and thermodynamic studies.

Goat β-lactoglobulin (GLG), lipocalin protein sharing high sequence similarity to bovine β-lactoglobulin (BLG), has been structurally and thermodynamically characterized. Two crystal forms of GLG have been obtained, trigonal (P3121) and orthorhombic (P21212), with unique molecular packing, not observed previously for BLG. In the trigonal structure, GLG molecules have EF-loop in closed conformation while in the orthorhombic structure, for the first time, symmetric and asymmetric dimers of β-lactoglobulin are observed simultaneously. It indicates that the opening or closing EF-loop does not occur in both subunits at the same time but might be sequential and cooperative. Comparison of GLG and BLG structures revealed presence of various conformers of EF and GH. ITC studies showed that at pH 7.5 GLG binds sodium dodecyl sulfate with Gibbs energy similar to BLG, however, with different contribution from enthalpic and entropic component. At pH 7.5 GLG forms dimers with dimerization constant Ka = 34.28 × 10(3) M(-1), significantly higher than observed for BLG. Similar mechanism of conformational changes and ligand binding indicates that GLG and BLG may play analogous biological role.

Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D1 and D2 receptor oligomerization

We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D2 receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C‐terminus of the dopamine D1 receptor (404EE405) on the receptors’ localization and their interaction. We studied receptor dimer formation using fluorescence resonance energy transfer. Receptor proteins were tagged with fluorescence proteins and expressed in HEK293 cells. The degree of D1–D2 receptor heterodimerization strongly depended on the number of Arg residues replaced by Ala in the ic3 of D2R, which may suggest that the indicated region of ic3 in D2R might be involved in interactions between two dopamine receptors. In addition, the subcellular localization of these receptors in cells expressing both receptors D1–cyan fluorescent protein, D2–yellow fluorescent protein, and various mutants was examined by confocal microscopy. Genetic manipulations of the Arg‐rich epitope induced alterations in the localization of the resulting receptor proteins, leading to the conclusion that this epitope is responsible for the cellular localization of the receptor. The lack of energy transfer between the genetic variants of yellow fluorescent protein‐tagged D2R and cyan fluorescent protein‐tagged D1R may result from differing localization of these proteins in the cell rather than from the possible role of the D2R basic domain in the mechanism of D1–D2 receptor heterodimerization. However, we find that the acidic epitope from the C‐terminus of the dopamine D1 receptor is engaged in the heterodimerization process.

The differences in binding 12-carbon aliphatic ligands by bovine beta-lactoglobulin isoform A and B studied by isothermal titration calorimetry and X-ray crystallography

Isoforms A (LGB‐A) and B (LGB‐B) of bovine lactoglobulin, the milk protein, differ in positions 64 (D↔G) and 118 (V↔A). Interactions of LGB‐A and LGB‐B with sodium dodecyl sulfate (SDS), dodecyltrimethylammonium chloride (DTAC) and lauric acid (LA), 12‐carbon ligands possessing differently charged polar groups, were investigated using isothermal titration calorimetry and X‐ray crystallography, to study the proton linkage phenomenon and to distinguish between effects related to different isoforms and different ligand properties. The determined values of ΔS and ΔH revealed that for all ligands, binding is entropically driven. The contribution from enthalpy change is lower and shows strong dependence on type of buffer that indicates proton release from the protein varying with protein isoform and ligand type and involvement of LA and Asp64 (in isoform A) in this process. The ligand affinities for both isoforms were arranged in the same order, DTAC < LA < SDS, and were systematically lower for variant B. The entropy change of the complexation process was always higher for isoform A, but these values were compensated by changes in enthalpy, resulting in almost identical ΔG for complexes of both isoforms. The determined crystal structures showed that substitution in positions 64 and 118 did not influence the overall structure of LGB complexes. The chemical character of the ligand polar group did not affect the position of its aliphatic chain in protein β‐barrel, indicating a major role of hydrophobic interactions in ligand binding that prevailed even with the repulsion between positively charged DTAC and lysine residues located at binding site entrance. Copyright

Temperature-induced conformational changes within the regulatory loops L1 -L2-LA of the HtrA heat-shock protease from Escherichia coli

The present investigation was undertaken to characterize mechanism of thermal activation of serine protease HtrA (DegP) from Escherichia coli. We monitored the temperature-induced structural changes within the regulatory loops L1, L2 and LA using a set of single-Trp HtrA mutants. The accessibility of each Trp residue to aqueous medium at temperature range 25-45 degrees C was assessed by steady-state fluorescence quenching using acrylamide and these results in combination with mean fluorescence lifetimes (tau) and wavelength emission maxima (lambda(em)max) were correlated with the induction of the HtrA proteolytic activity. Generally the temperature shift caused better exposure of Trps to the quencher; although, each of the loops was affected differently. The LA loop seemed to be the most prone to temperature-induced conformational changes and a significant opening of its structure was observed even at the lowest temperatures tested (25-30 degrees C). To the contrary, the L1 loop, containing the active site serine, remained relatively unchanged up to 40 degrees C. The L2 loop was the most exposed element and showed the most pronounced changes at temperatures exceeding 35 degrees C. Summing up, the HtrA structure appears to open gradually, parallel to the gradual increase of its proteolytic activity.

Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by dynamic light scattering and time-resolved fluorescence anisotropy methods.

Cyclic AMP receptor protein (CRP) regulates the expression of more than 100 genes in Escherichia coli when complexed with cyclic AMP. Dynamic light scattering (DLS) and fluorescence decay anisotropy measurements of CRP were performed in solution, in the absence and presence of cAMP. We have also measured the effect of DNA sequences, including lac and gal promoter sequences, on the shape of CRP-DNA complexes. DLS measurements show that upon cAMP binding at low nucleotide concentration, the Stokes radius decreases from the value of 2.8 nm for apo-CRP to the value of 2.7 nm. At higher cAMP concentration, only a very small further decrease was detected. Fluorescence anisotropy decay measurements, with the use of CRP labeled at Cys-178 with 1,5-I-AENS, indicate that apo-CRP exhibits two rotational correlation times. The longer time, θ1 = 23.3 ns, corresponds to the overall motion of the protein, and the shorter time, θ2 = 1.4 ns, exhibits segmental mobility of the C-terminal domain of CRP. Binding of cAMP into CRP induced substantial increase of θ1 to the value of 30.7 ns, whereas θ2 remained unchanged. The DLS measurements indicate that the binding of CRP into a fragment of DNA possessing a sequence of lac promoter induces a larger increase in the Stokes radius of lac-CRP complex than in case of gal-CRP complex. Similarly, a higher change was detected in rotational correlation time, θ1, in the case of lac-CRP complex than in case of gal-CRP. Because the lac and gal promoters are characteristic for the two different classes of CRP-dependent promoters, one can expect that the observed differences in lac-CRP and gal-CRP complexes are important in activation of transcription in Escherichia coli.