Zygmunt Wasylewski

The concept that G protein-coupled receptors (GPCRs) function as oligomers has been widely accepted, however, different methodologies often used to study the phenomenon of GPCR interactions do not allow, as yet, for any generalization as to whether di- or oligomers are formed constitutively or are ligand-promoted. Here, we report on the use of three independent biophysical approaches based on the Förster resonance energy transfer to study the adenosine A2A and serotonin 5-HT1A receptor (tagged with derivatives of green fluorescence protein, CFP - fluorescence donor and YFP - fluorescence acceptor) homodimerization in the plasma membrane of transiently transfected HEK 293 cell line. Homodimers of A2A and 5-HT1A receptors are formed constitutively, however, specific ligands regulate the degree of these interactions: agonists (CGS 21680 and 8-OH-DPAT, respectively) further enhanced while antagonists (SCH 58216 and methysergide) diminished the dimer formation. Although the acceptor photobleaching with the use of confocal microscopy as well as the fluorescence lifetime microscopy gave similar results, we strongly recommend the latter technique as a highly sensitive and quantitative approach to that kind of the study. The additional proof of specificity of the observed results is provided by the studies of interaction of adenosine A2A and serotonin 5-HT1A receptors with the alpha subunits of G proteins. The A2A receptor interacted with G alpha s and 5-HT1A receptor - with G alpha i, while physical interaction of these receptors with no appropriate alpha subunits partners (A2A-G alpha i and 5HT1A-G alpha s) has not been observed, despite the identical level of overexpression of proteins in all studied combinations.

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. We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule. The results presented provide evidence for the occurrence of a long‐distance conformational signal transduction within the protein from the C‐terminal DNA‐binding domain to the N‐terminal domain of CRP. This conformational signal transmission depends on the promoter sequence. We also used the stopped‐flow and Förster resonance energy transfer between labeled Cys178 of CRP and fluorescently labeled DNA sequences to study the kinetics of DNA–CRP interactions. The results thus obtained lead to the conclusion that CRP can exist in several conformational states and that their distribution is affected by binding of both the cAMP and of specific DNA sequences.

Tetracycline repressor (TetR), which constitutes the most common mechanism of bacterial resistance to an antibiotic, is a homodimeric protein composed of two identical subunits, each of which contains a domain possessing a helix–turn–helix motif and a domain responsible for binding tetracycline. Binding of tetracycline in the protein pocket is accompanied by conformational changes in TetR, which abolish the specific interaction between the protein and DNA. Differential scanning calorimetry (DSC) and CD measurements, performed at pH 8.0, were used to observe the thermal denaturation of TetR in the absence and presence of tetracycline. The DSC results show that, in the absence of tetracycline, the thermally induced transitions of TetR can be described as an irreversible process, strongly dependent on scan rate and indicating that the protein denaturation is under kinetic control described by the simple kinetic scheme: , where k is a first‐order kinetic constant, N is the native state, and D is the denatured state. On the other hand, analysis of the scan rate effect on the transitions of TetR in the presence of tetracycline shows that thermal unfolding of the protein can be described by the two‐state model: . In the proposed model, TetR in the presence of tetracycline undergoes co‐operative unfolding, characterized by an enthalpy change (ΔH_cal = 1067 kJ·mol⁻¹) and an entropy change (ΔS = 3.1 kJ·mol⁻¹).

The cyclic AMP receptor protein (CRP) regulates the expression of many genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. In this...

CAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady‐state and time‐resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5‐I‐AEDANS attached to Cys178. Binding of cAMP in the CRP–(cAMP)₂ complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP–(cAMP)₄ complex alters the surroundings of Trp85. Time‐resolved anisotropy measurements indicated that cAMP binding in the CRP–(cAMP)₂ complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP–(cAMP)₂ complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 Å in apo‐CRP to 18.7 Å in the CRP–(cAMP)₂ complex. The binding of cAMP in the CRP–(cAMP)₄ complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP–DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to ≈ 20 Å. The spectral changes observed provide new structural information about the cAMP‐induced allosteric activation of the protein.

Cyclic AMP receptor protein (CRP) regulates the expression of more than 100 genes in Escherichia coliwhen complexed with cyclic AMP. Dynamic light scattering (DLS) and fluorescence decay anisotropy...

Steady-state quenching and time-resolved fluorescence measurements of L-tryptophan binding to the tryptophan-free mutant W19/99F of the tryptophan repressor of Escherichia colihave been used to...

Kinetic measurements of denaturation and renaturation of two mutants of cAMP receptor protein (CRP) at position 128, namely Ser→Ala and Ser→Pro, were performed in order to assess changes introduced...

CAMP receptor protein (CRP) is involved in regulation of expression of several genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two distinct structural domains. The mechanism of the biological activity of the protein may involve the interaction between the subunits and domains. In order to determine the interaction between the subunits or domains of CRP, we have studied the reversible denaturation of the protein by guanidine hydrochloride. The unfolding and refolding kinetics of CRP was monitored using stopped‐flow fluorescence spectroscopy at 20°C and pH 7.9. The results of CRP denaturation indicate that the transition can be described by a three‐state model: (CRP_native)₂⇔ 2 (CRP_native) ⇔ 2 (CRP_denatured). The faster process, characterized by the relaxation time τ₂= 80 ± 3 ms, corresponds to the dissociation of CRP dimer into monomers. The slower process has the relaxation time τ₁, = 1.9 ± 0.1 s and corresponds to the cooperative unfolding of CRP monomer. The free energy change in the absence of denaturant upon CRP dissociation is G_dis⁰= 46.9 ± 2.5 kJ/mol and for monomer unfolding G_unf⁰, = 30.9 ± 1.3 kJ/mol. The thermal unfolding of CRP was studied by circular dichroism and fluorescence spectroscopy at various guanidine hydrochloride concentrations. It has been found that the native protein is maximally stable at about 21 = 0.3 °C and is denatured upon heating and cooling from this temperature. The apparent free energy change for CRP unfolding at 21 °C is equal to 30.5 ± 20.4 kJ/mol and the apparent spccific heat change is equal to c_p,app= 10.7 ± 0.7 kJ/mol⁻¹ K⁻¹. The predicted values of cold denaturation midpoint is equal to t'_G=–18.8 ± 1.5°C and for high‐temperature transition t_G, = 63.1 ± 1.5°C. The predicted midpoint of high‐temperature unfolding transition is about the same as determined experimentally.