| 1. |
- Andrésen, Cecilia, et al.
(författare)
-
Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
- 2010
-
Ingår i: Protein Science. - : Cold Spring Harbor Laboratory Press. - 0961-8368 .- 1469-896X. ; 19:4, s. 680-692
-
Tidskriftsartikel (refereegranskat)abstract
- The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.
|
|
| 2. |
|
|
| 3. |
- Jarl, Anngelica, et al.
(författare)
-
AEDANS labeled MexR variants and their DNA interaction
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- MexR is a transcriptional repressor of the multidrug efflux-pump operon in the bacterial pathogen Pseudomonas aeruginosa, and thus, malfunction in the DNA-binding of the MexR protein leads to multidrug resistance (Poole et al., 1996). lt has been shown that mutations in the MexR protein lead to lost DNA binding ability (Saito et al., 2003). X-ray studies of the MexR protein (Lim et al., 2002) show that the protein contains a winged helix-turn-helix motif and this type of motif is known to be involved in DNA-binding (Gajiwala & Burley, 2000), but no exact DNA-binding sequence in MexR has yet been determined.This study attempts to identify regions in the MexR protein involved in DNA-binding using a molecular probe as mapping tool. Three cysteines naturally occur in the MexR protein, located in positions 30, 62 and 138, respectively, and we have used two of them to map the DNA-binding site. Preliminary results show that the fluorescence of an TAEDANS probe attached to cys 62 is affected by DNA-binding, while a probe at cys 30 is not affected. Further studies are required to analyse the implications of these results.
|
|
| 4. |
- Jarl, Anngelica, 1977-
(författare)
-
Molecular probes as protein analysis tools in biotechnology
- 2008
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein-labeling today is a work of art, in vivo studies of proteins or other molecules can easily be performed, and the movement of the labeled molecule can be followed in real time. Labeling in vitro gives enormous amount of data in labs all over the world on a daily basis, where protein-protein, protein-DNA or other interactions are studied. Folding and unfolding events can be monitored wi th labels reporting on local or global environmental changes in a protein. The use of labeling seems endless, but in this thesis I have chosen to focus on two labeling techniques: spin-labeling and fluorescence labeling. Applying these techniques on protein-protein and protein-DNA interactions has resulted in better understanding of protein folding and function.Chaperonin function at elevated temperaturesThe model protein HCA II (259 amino acids) mainly consisting of a large 10 stranded ß-sheet with a topological breakpoint between strands 6 and 7. Two residues, adjacent in the folded structure and located at each side of this breakpoint, were used in a site-directed-spin-labeling (SDSL) experiment. The aim was to elucidate what happens at the breakpoint when the protein interacts with the chaperonin GroEL at elevated temperatures. The chaperonin GroEL is a 60 kDa protein known to aid protein folding in the cell. By probing the model protein, HCA II, we have shown that this chaperone can stretch its substrate and release it for a new refolding opportunity.MexR protein interaction with DNAMexR is a 147 amino acid protein dimer involved in transcriptional repression of the multidrug efflux operon MexAB-OprM in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Malfunction in MexR results in multidrug resistant bacteria resistant to therapeutic strategies. Site-specific fluorescence-labeling of MexR has been investigated as a means to provide a new strategy for localising DNA binding and quantifyi ng DNA affinity. ANS fluorescence of the MexR protein in the absence and presence of DNA, together with a range of biophysical measurements, has provided a new view on how MexR could be regulated by small molecule binding, and thus sheds new light on its functionality in gene repression.
|
|
| 5. |
|
|
| 6. |
- Owenius, Rikard, et al.
(författare)
-
GroEL-induced stretching of a substrate protein: An EPR/SDSL study in BIOPHYSICAL JOURNAL, vol 88, issue 1, pp 562A-562A
- 2005
-
Ingår i: BIOPHYSICAL JOURNAL. - : Elsevier (Cell Press) / Biophysical Society. ; , s. 562A-562A
-
Konferensbidrag (refereegranskat)abstract
- The Hsp60-type chaperonin GroEL assists in the folding of the enzyme Human Carbonic Anhydrase II (HCA II) and protects it from aggregation.It is still a controversy whether the action of GroEL is an active or passive process. Single- and double-cysteine mutants were specificallyspin labeled at a topological breakpoint in the β-core of HCA II. X-band electron paramagnetic resonance (EPR) was used at physiologicaltemperatures to monitor the GroEL-induced structural changes in this region of HCA II. Inter-residue distance calculations based on dipolarinteraction show that the proximity of the labeled positions F147 and K213 in the native state of HCA II is ~11±2 Å, and that it is virtuallyintact in the thermally-induced molten-globule state that binds to GroEL. However, upon interaction with GroEL a spin-spin distance increaseto ~22±3 Å indicates a conformational change in HCA II that is part of the GroEL-induced substrate stretch that enables structural rearrangementof a misfolded substrate protein.
|
|
| 7. |
- Owenius, Rikard, et al.
(författare)
-
GroEL-induced topological dislocation of a substrate protein β-sheet core : a solution EPR spin–spin distance study
- 2010
-
Ingår i: Journal of chemical biology. - : Springer Science and Business Media LLC. - 1864-6158 .- 1864-6166. ; 3:3, s. 127-39
-
Tidskriftsartikel (refereegranskat)abstract
- The Hsp60-type chaperonin GroEL assists in the folding of the enzyme human carbonic anhydrase II (HCA II) and protects it from aggregation. This study was aimed to monitor conformational rearrangement of the substrate protein during the initial GroEL capture (in the absence of ATP) of the thermally unfolded HCA II molten-globule. Single- and double-cysteine mutants were specifically spin-labeled at a topological breakpoint in the β-sheet rich core of HCA II, where the dominating antiparallel β-sheet is broken and β-strands 6 and 7 are parallel. Electron paramagnetic resonance (EPR) was used to monitor the GroEL-induced structural changes in this region of HCA II during thermal denaturation. Both qualitative analysis of the EPR spectra and refined inter-residue distance calculations based on magnetic dipolar interaction show that the spin-labeled positions F147C and K213C are in proximity in the native state of HCA II at 20 °C (as close as ∼8 Å), and that this local structure is virtually intact in the thermally induced molten-globule state that binds to GroEL. In the absence of GroEL, the molten globule of HCA II irreversibly aggregates. In contrast, a substantial increase in spin–spin distance (up to >20 Å) was observed within minutes, upon interaction with GroEL (at 50 and 60 °C), which demonstrates a GroEL-induced conformational change in HCA II. The GroEL binding-induced disentanglement of the substrate protein core at the topological break-point is likely a key event for rearrangement of this potent aggregation initiation site, and hence, this conformational change averts HCA II misfolding.
|
|
