| 1. |
- Bergström, Fredrik, et al.
(author)
-
Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology.
- 2002
-
In: Journal of the American Chemical Society. - 0002-7863 .- 1520-5126. ; 124:2, s. 196-204
-
Journal article (peer-reviewed)abstract
- A ground-state dimer (denoted D(I)) exhibiting a strong absorption maximum at 477 nm (epsilon = 97 000 M(-1)cm(-1)) can form between adjacent BODIPY groups attached to mutant forms of the protein, plasminogen activator inhibitor type 1 (PAI-1). No fluorescence from excited D(I) was detected. A locally high concentration of BODIPY groups was also achieved by doping lipid phases (micelles, vesicles) with BODIPY-labeled lipids. In addition to an absorption band located at about 480 nm, a new weak absorption band is also observed at ca. 570 nm. Both bands are ascribed to the formation of BODIPY dimers of different conformation (D(I) and D(II)). Contrary to D(I) in PAI-1, the D(II) aggregates absorbing at 570 nm are emitting light observed as a broad band centered at about 630 nm. The integrated absorption band of D(I) is about twice that of the monomer, which is compatible with exciton coupling within a dimer. The Förster radius of electronic energy transfer between a BODIPY excited monomer and the ground-state dimer (D(I)()) is 57 +/- 2 A. A simple model of exciton coupling suggests that in D(I) two BODIPY groups are stacked on top of each other in a sandwich-like configuration with parallel electronic transition dipoles. For D(II) the model suggests that the S(0) --> S(1) transition dipoles are colinear. An explanation for the previously reported (J. Am. Chem. Soc. 1994, 116, 7801) exceptional light spectroscopic properties of BODIPY is also presented. These are ascribed to the extraordinary electric properties of the BODIPY chromophore. First, changes of the permanent electric dipole moment (Delta(mu) approximately -0.05 D) and polarizability (-26 x 10(-40) C m(2) V(-1)) between the ground and the first excited states are small. Second, the S(0) <--> S(1) electronic transition dipole moments are perpendicular to Delta(mu).
|
|
| 2. |
- Bergström, Fredrik, et al.
(author)
-
Dimers of Dipyrrometheneboron Difluoride (BODIPY) with Light Spectroscopic Applications in Chemistry and Biology
- 2002
-
In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 124:2, s. 196-204
-
Journal article (peer-reviewed)abstract
- A ground-state dimer (denoted DI) exhibiting a strong absorption maximum at 477 nm ( = 97 000 M-1cm-1) can form between adjacent BODIPY groups attached to mutant forms of the protein, plasminogen activator inhibitor type 1 (PAI-1). No fluorescence from excited DI was detected. A locally high concentration of BODIPY groups was also achieved by doping lipid phases (micelles, vesicles) with BODIPY-labeled lipids. In addition to an absorption band located at about 480 nm, a new weak absorption band is also observed at ca. 570 nm. Both bands are ascribed to the formation of BODIPY dimers of different conformation (DI and DII). Contrary to DI in PAI-1, the DII aggregates absorbing at 570 nm are emitting light observed as a broad band centered at about 630 nm. The integrated absorption band of DI is about twice that of the monomer, which is compatible with exciton coupling within a dimer. The Förster radius of electronic energy transfer between a BODIPY excited monomer and the ground-state dimer (DI) is 57 ± 2 Å. A simple model of exciton coupling suggests that in DI two BODIPY groups are stacked on top of each other in a sandwich-like configuration with parallel electronic transition dipoles. For DII the model suggests that the S0 S1 transition dipoles are collinear. An explanation for the previously reported (J. Am. Chem. Soc. 1994, 116, 7801) exceptional light spectroscopic properties of BODIPY is also presented. These are ascribed to the extraordinary electric properties of the BODIPY chromophore. First, changes of the permanent electric dipole moment ( -0.05 D) and polarizability (-26 × 10-40 C m2 V-1) between the ground and the first excited states are small. Second, the S0 S1 electronic transition dipole moments are perpendicular to .
|
|
| 3. |
- Fa, M, et al.
(author)
-
Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.
- 2000
-
In: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 267:12, s. 3729-34
-
Journal article (peer-reviewed)abstract
- The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.
|
|
| 4. |
- Fa, Ming, et al.
(author)
-
The structure of a serpin–protease complex revealed by intramolecular distance measurements using donor–donor energy migration and mapping of interaction sites
- 2000
-
In: Structure. ; 8:4, s. 397-405
-
Journal article (peer-reviewed)abstract
- Background: The inhibitors that belong to the serpin family are widely distributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-centre cleavage, loop insertion and protease translocation, but different models of the serpin–protease complex have been proposed. In the absence of a spatial structure of a serpin–protease complex, a detailed understanding of serpin inhibition and the character of the virtually irreversible complex have remained controversial.Results: We used a recently developed method for making precise distance measurements, based on donor–donor energy migration (DDEM), to accurately triangulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PAI-1). The distances from residue 344 (P3) in the reactive-centre loop of PAI-1 to residues 185, 266, 313 and 347 (P1′) were determined. Modelling of the complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the reactive-centre loop fully inserted into β sheet A. To validate the model, seven single cysteine substitution mutants of PAI-1 were used to map sites of protease–inhibitor interaction by fluorescence depolarisation measurements of fluorophores attached to these residues and cross-linking using a sulphydryl-specific cross-linker.Conclusions: The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.
|
|
| 5. |
- Hosokawa, K, et al.
(author)
-
Dominant expression and distribution of oestrogen receptor beta over oestrogen receptor alpha in the human corpus luteum
- 2001
-
In: Molecular human reproduction. - : Oxford University Press (OUP). - 1360-9947 .- 1460-2407. ; 7:2, s. 137-145
-
Journal article (peer-reviewed)abstract
- To investigate the potential importance of oestrogen as a local regulator of human corpus luteum function, the mRNA expression pattern and cellular localization of oestrogen receptors (ERs), ER-alpha and ER-beta, were studied in corpora lutea grouped according to age, where days 2-5 post-LH rise were designated as the early luteal phase, days 6-10 as mid-luteal and days 11-14 as the late luteal phase respectively. Northern blot analysis using an ER-beta probe in samples from whole ovarian tissue and isolated corpora lutea, revealed a major band at 7.5 kb and several minor bands between 4-10 kb, while no signals for ER-alpha mRNA were obtained. However, using a semi-quantitative reverse transcription-polymerase chain reaction followed by Southern blotting, ER-beta mRNA levels were found to be 63% lower (P: < 0.05, n = 39) in the mid-luteal phase compared with the early luteal phase, while ER-alpha mRNA expression showed no statistical differences between the different age groups. Using in-situ hybridization, ER-beta mRNA expression was localized to the steroidogenic luteal cells as well as perivascular cells and fibroblasts in the corpus luteum. Immunohistochemistry confirmed the localization of ER-beta protein, but no clear staining of luteal cells was found using antibodies against ER-alpha. Collectively, the findings of low to moderate expression of ER-beta mRNA and protein in the steroidogenic cells, and also in vascular endothelial cells of the corpus luteum, as opposed to diminutive amounts of ER-alpha mRNA, suggest that oestrogen activity is primarily transduced via ER-beta in the human corpus luteum.
|
|
| 6. |
- Hägglund, A C, et al.
(author)
-
Stromelysin-3 is induced in mouse ovarian follicles undergoing hormonally controlled apoptosis, but this metalloproteinase is not required for follicular atresia.
- 2001
-
In: Biology of Reproduction. - 0006-3363 .- 1529-7268. ; 64:2, s. 457-63
-
Journal article (peer-reviewed)abstract
- Apoptotic processes are often associated with an intense proteolytic remodeling of the extracellular matrix (ECM). Proteolytic degradation of the ECM can also be a signal that induces apoptosis. Here, we have investigated the expression pattern and functional role of the matrix metalloproteinase stromelysin-3 in follicular atresia. Twenty-four hours after the treatment of immature female mice with a low dose of eCG, both apoptosis and the stromelysin-3 mRNA expression were suppressed approximately threefold. However, the initial suppression of apoptosis and stromelysin-3 expression was followed by a time-dependent increase, and 96 h after eCG treatment, the levels were similar to those of untreated control mice. In 15- to 16-day-old juvenile mice, the ovary consisted of relatively undeveloped follicles, and almost no apoptosis and only low stromelysin-3 mRNA expression were observed. However, at the age of 21 days, when several antral follicles were present, a fivefold induction in both apoptosis and stromelysin-3 mRNA expression was detected. For both models, in situ analysis revealed that the expression of stromelysin-3 mRNA was localized to the granulosa cells of atretic follicles. To address the functional role of stromelysin-3 in follicular atresia, stromelysin-3-deficient mice were studied. However, no difference in the pattern of apoptotic DNA fragmentation and no apparent morphological differences were observed when ovaries from wild-type and stromelysin-3-deficient mice were compared. Taken together, our data indicate that stromelysin-3 is induced during follicular atresia, but that this protease is not obligatory for initiation or completion of the atretic process.
|
|
| 7. |
- Hägglöf, Peter, et al.
(author)
-
The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted
- 2004
-
In: Journal of Molecular Biology. - London : Academic Press. - 0022-2836 .- 1089-8638. ; 335:3, s. 823-832
-
Journal article (peer-reviewed)abstract
- Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1′ residues acting as a “bait” for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into β-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor–donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in β-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into β-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.
|
|
| 8. |
- Isaksson, Mikael, et al.
(author)
-
An environmental-sensitive BODIPY®-derivative with bioapplication : spectral and photophysical properties
- 2003
-
In: Journal of Fluorescence. - 1053-0509 .- 1573-4994. ; 13:5, s. 379-84
-
Journal article (peer-reviewed)abstract
- A previously synthesised derivative of BODIPY aimed for sulfhydryl specific labelling of cysteine residues in proteins was studied. The spectral and photophysical properties of this derivative, N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl) iodoacetamide (NBDY) were characterised, and found to be considerably different from those of commonly used derivatives of BODIPY, e.g. N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide. The absorption and fluorescence spectra, as well as fluorescence lifetimes and quantum yields of NBDY are quite sensitive to solvent properties. The fluorescence is effectively quenched by I– when NBDY is free in water or attached to Cys in different mutants of plasminogen activator inhibitor type 2 (PAI-2). A ground-state dimer forms when two NBDY groups are closely spaced in plasminogen activator inhibitor type 1 (PAI-1).
|
|
| 9. |
- Isaksson, Mikael, et al.
(author)
-
Partial donor-donor energy migration (PDDEM) : a novel fluorescence method for internal protein distance measurements
- 2004
-
In: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 6:11, s. 3001-3008
-
Journal article (peer-reviewed)abstract
- We show that the photophysics of chemically identical but photophysically non-identical fluorescent pairs can be used for measuring distances within proteins. For this purpose, the theory of partial donor-donor energy migration (PDDEM, S. Kalinin, J. G. Molotkovsky and L. B.-Angstrom. Johansson, Spectrochim. Acta, Part A, 2002, 58, 1057-1097) was applied for distance measurements between BODIPY groups covalently linked to cystein residues in plasminogen activator inhibitor of type 2 (PAI-2). Two sulfhydryl specific derivatives of BODIPY were used namely: N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl) iodoacetamide and N-(4.4-difluoro-5.7-ditriethyl-4-bona-3a,4a-diaza-s-indacene-3-yl) methyl iodoacetamide. To determine distances, the time-resolved fluorescence relaxation for two singly labelled forms of PAI-2, as well as the corresponding doubly labelled protein were combined and analysed in a global manner. Fluorescence depolarisation experiments on the labelled mutants were also analysed. The distances determined by PDDEM were in good agreement to those obtained from donor-donor energy migration (DDEM) experiments and structural data on PAI-2. The PDDEM approach allows for the use of very different fluorescent probes, which enables wide range of distances to be measured. The PDDEM model also provides a rational explanation to why previous observations of polyfluorophore-labelled proteins exhibit a shorter average fluorescence lifetime compared to the arithmetic average of lifetimes obtained for the corresponding single labelled proteins.
|
|
| 10. |
|
|
