Structure-based design approach to rational site-directed mutagenesis of β-lactoglobulin.

Recombinant proteins play an important role in medicine and have diverse applications in industrial biotechnology. Lactoglobulin has shown great potential for use in targeted drug delivery and body fluid detoxification because of its ability to bind a variety of molecules. In order to modify the biophysical properties of β-lactoglobulin, a series of single-site mutations were designed using a structure-based approach.

PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.

DNA lesions induce recruitment and accumulation of various repair factors, resulting in formation of discrete nuclear foci. Using superresolution fluorescence microscopy as well as live cell and quantitative imaging, we demonstrate that X-ray repair cross-complementing protein 1 (XRCC1), a key factor in single-strand break and base excision repair, is recruited into nuclear bodies formed in response to replication-related single-strand breaks. Intriguingly, these bodies are assembled immediately in the vicinity of these breaks and never fully colocalize with replication foci.

The engineered β-lactoglobulin with complementarity to the chlorpromazine chiral conformers.

Chlorpromazine (CPZ) is a phenothiazine acting as dopamine antagonist. Aside from application in schizophrenia therapy, chlorpromazine is found to be a putative inhibitor of proteins involved in cancers, heritable autism disorder and prion diseases. Four new β-lactoglobulin variants with double or triple substitutions: I56F/L39A, F105L/L39A, I56F/L39A/M107F or F105L/L39A/M107F changing the shape of the binding pocket were produced and their chlorpromazine binding properties have been investigated by X-ray crystallography, circular dichroism, isothermal titration calorimetry and thermophoresis.

Bradykinin B2 and dopamine D2 receptors form a functional dimer.

In recent years a wide range of studies have shown that G protein-coupled receptors modulate a variety of cell functions through the formation of dimers. For instance, there is growing evidence for the dimerization of bradykinin or dopamine receptors, both as homodimers and heterodimers. A discovery of direct interactions of angiotensin II receptors with bradykinin 2 receptor (B2R) or dopamine D2 (D2R) receptor has led to a hypothesis on a potential dimerization between two latter receptors.

The role of cholesterol and sphingolipids in the dopamine D receptor and G protein distribution in the plasma membrane.

G proteins are peripheral membrane proteins which interact with the inner side of the plasma membrane and form part of the signalling cascade activated by G protein-coupled receptors (GPCRs). Since many signalling proteins do not appear to be homogeneously distributed on the cell surface, they associate in particular membrane regions containing specific lipids. Therefore, protein-lipid interactions play a pivotal role in cell signalling.

Engineered β-Lactoglobulin Produced in E. coli: Purification, Biophysical and Structural Characterisation.

Functional recombinant bovine β-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suitable for biochemical, biophysical and structural studies to be obtained.

New insights into the model of dopamine D1 receptor and G-proteins interactions.

The details of the interaction between G-proteins and the GPCRs have been subjected to extensive investigation with structural and functional assays, but still many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered. In the context of current structural data we investigated interactions of dopamine D1 receptor with cognate G-proteins (Gαs) in living cells, emphasizing the prevalence of preassembled D1-G-protein complexes. We also tested the effect of D1 receptor presence on the dynamics of Gαs and Gαi3 in the cellular plasma membrane.

Role of silent polymorphisms within the dopamine D1 receptor associated with schizophrenia on D1-D2 receptor hetero-dimerization.

Within the coding region of the dopamine D(1) receptor (D(1)R), two synonymous polymorphisms, D(1)R(G198A) and D(1)R(G1263), have been identified and postulated to correlate with the schizophrenia phenotype. Binding studies revealed that the density of these genetic variants was much lower than the density of wild type D(1)R in the human embryonic kidney (HEK) 293 cell line, used as a model system. From the data obtained using MFOLD software it is apparent that the G198A mutation has a greater impact on the secondary structure of the mRNA, which may affect its translation.

Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli.

Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-terminal cAMP-binding domain to the C-terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N-terminal domain of the protein.