Designed fluorescent probes reveal interactions between amyloid-beta(1-40) peptides and GM1 gangliosides in micelles and lipid vesicles.

A hallmark of the common Alzheimer's disease (AD) is the pathological conversion of its amphiphatic amyloid-beta (Abeta) peptide into neurotoxic aggregates. In AD patients, these aggregates are often found to be tightly associated with neuronal G(M1) ganglioside lipids, suggesting an involvement of G(M1) not only in aggregate formation but also in neurotoxic events. Significant interactions were found between micelles made of newly synthesized fluorescent G(M1) gangliosides labeled in the polar headgroup or the hydrophobic chain and Abeta(1-40) peptide labeled with a BODIPY-FL-C1 fluorophore at positions 12 and 26, respectively. From an analysis of energy transfer between the different fluorescence labels and their location in the molecules, we were able to place the Abeta peptide inside G(M1) micelles, close to the hydrophobic-hydrophilic interface. Large unilamellar vesicles composed of a raftlike G(M1)/bSM/cholesterol lipid composition doped with labeled G(M1) at various positions also interact with labeled Abeta peptide tagged to amino acids 2 or 26. A faster energy transfer was observed from the Abeta peptide to bilayers doped with 581/591-BODIPY-C(11)-G(M1) in the nonpolar part of the lipid compared with 581/591-BODIPY-C(5)-G(M1) residing in the polar headgroup. These data are compatible with a clustering process of G(M1) molecules, an effect that not only increases the Abeta peptide affinity, but also causes a pronounced Abeta peptide penetration deeper into the lipid membrane; all these factors are potentially involved in Abeta peptide aggregate formation due to an altered ganglioside metabolism found in AD patients.

Fluorescence spectroscopic properties analysed within the extended Förster theory with application to biomacromolecular systems.

The extended Förster theory (EFT) of electronic energy transport accounts for translational and rotational dynamics, which are neglected by the classical Förster theory (FT). EFT has been developed for electronic energy transfer within donor-acceptor pairs [Isaksson, et al, Phys. Chem. Chem. Phys., 9, 1941(2007)] and donor-donor pairs [Johansson, et al, J. Chem. Phys., 105, 10896 (1996); Norlin, et al, Phys. Chem. Chem. Phys., 10, 6962(2008)]. For donors that exhibit different or identical non-exponential fluorescence relaxation within a donor-donor pair, the process of reverberating energy migration is reversible to a higher or lower degree. Here the impact of the EFT has been studied with respect to its influence on fluorescence quantum yields, fluorescence lifetimes as well as depolarisation experiments. The FT predicts relative fluorescence quantum yields which usually agree with the EFT within experimental accuracy, however, substantial deviations occurs in the steady-state and in particular the time-resolved depolarisation data.

Extended Förster theory for determining intraprotein distances. Part III. Partial donor-donor energy migration among reorienting fluorophores.

An extended Förster theory (EFT) is derived and outlined for electronic energy migration between two fluorescent molecules which are chemically identical, but photophysically non-identical. These molecules exhibit identical absorption and fluorescence spectra, while their fluorescence lifetimes differ. The latter means that the excitation probability becomes irreversible. Unlike the case of equal lifetimes, which is often referred to as, donor-donor energy migration (DDEM), the observed fluorescence relaxation is then no longer invariant to the energy migration process. To distinguish, the present case is therefore referred to as partial donor-donor energy migration (PDDEM). The EFT of PPDEM is described by a stochastic master equation (SME), which has been derived from the stochastic Liouville equation (SLE) of motion. The SME accounts for the reorienting as well as the translational motions of the interacting chromophores. Synthetic fluorescence lifetime and depolarisation data that mimics time-correlated single photon counting experiments have been generated and re-analysed. The rates of reorientation, as well as the orientational configurations of the interacting D-groups were examined. Moreover the EFT of PPDEM overcomes the classical "kappa(2)-problem" and the frequently applied approximation of kappa(2) = 2/3 in the data analyses. An outline for the analyses of fluorescence lifetime and depolarisation data is also given, which might prove applicable to structural studies of D-labelled macromolecules, e.g. proteins. The EFT presented here brings the analyses of PDDEM data to the same level of molecular detail as that used in ESR- and NMR-spectroscopy.

[New fluorescent bichromophoric probes for studying mechanisms of donor-donor energy migration (DDEM)]. / Novye fluorestsentnye bikhromofornye zondy dlia issledovaniia mekhanizmov donor-donornoi migratsii énergii (DDEM).

Three fluorescent probes were synthesized for studying the excitation energy migration between two identical fluorophores. Each probe has two identical fluorescent groups (dansyl, 7-nitrobenz-2-oxa-1,3-diazole-4-yl, or fluoresceinyl) linked by the rigid bis-(8-aminooctyl)amide of 4,4'-biphenyldicarbonic acid or flexible dotriacontanedioic acid spacer, which enables the intramolecular energy migration through the distance of 3.2-3.5 nm.

The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome.

The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058-9, U2506 and U2584-5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer.

Donor-donor energy migration (DDEM) as a tool for studying aggregation in lipid phases.

A BODIPY-labelled sulfatide (N-(BODIPY-FL-pentanoyl)-galactosylcerebroside-sulfate, hereafter abbreviated as BD-Sulfatide) was solubilised at different concentrations in lipid vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Time-correlated single photon counting experiments show that the fluorescence relaxation is mono-exponential (with a lifetime of 6.5 ns) at molar ratios of BD-Sulfatide: DOPC that are less than 1:100. The fluorescence steady-state anisotropy decreases monotonously at molar ratios smaller than 1:1000, which is compatible with donor-donor energy migration (DDEM) among the BODIPY groups. A model that assumes DDEM across the lipid bilayers, as well as in their planes, was used to analyse the time-resolved fluorescence anisotropy. Only two parameters appear in the model namely: the bilayer thickness (d) and the average number density (C2) distribution of BD-Sulfatide in the lipid bilayers. The extracted d-values vary between 35 and 40 A, which is about the reported thickness of a bilayer of DOPC (38 A). Hence, the BODIPY groups are preferentially located in the water-lipid interface. At low concentration the experimental C2-values and those independently calculated are in good agreement, while the experimental values gradually become lower with increasing BD-Sulfatide concentration. These results are compatible with an aggregation of the sulfatides and self-quenching of BODIPY, which is clearly established at higher concentrations of the BD-Sulfatide.

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.

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.

The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor-donor energy migration and mapping of interaction sites.

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 beta 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.

The use of site-directed fluorophore labeling and donor-donor energy migration to investigate solution structure and dynamics in proteins.

The use of molecular genetics for introducing fluorescent molecules enables the use of donor-donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor-donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the C(alpha)-atoms of the positions V106C-H185C, H185C-M266C, and M266C-V106C were 60.9, 30.8, and 55.1 A, respectively. These are in good agreement with the distances of 54 +/- 4, 38 +/- 3, and 55 +/- 3 A, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the C(alpha)-atoms physically cannot coincide, there is a reasonable agreement between the methods.

New fluorescent cholesterol analogs as membrane probes.

New fluorescent cholesterol analogs, (22E, 20R)-3beta-hydroxy-23-(9-anthryl)-24-norchola-5,22-die ne (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synthesized, their spectral and membrane properties were characterized. The probes bear a 9-anthrylvinyl (AV) group instead of C22-C27 segment of the cholesterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, the force-area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N-stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35-40% more cross-sectional area than cholesterol at surface pressures below film collapse (0-22 mN/m); whereas S-AV-Ch occupies nearly the same molecular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fraction of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (lambda 386, 368, 350 and 256 nm) and fluorescence (lambda 412-435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or liquid-crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and decreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which show also, in accordance with monolayer data, that S-AV-Ch is better accommodated in POPC-cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle production. Detergent-solubilization studies involving 'raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that of R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV-labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies.

Donor-donor energy migration for determining intramolecular distances in proteins: I. Application of a model to the latent plasminogen activator inhibitor-1 (PAI-1).

A new fluorescence spectroscopic method is presented for determining intramolecular and intermolecular distances in proteins and protein complexes, respectively. The method circumvents the general problem of achieving specific labeling with two different chromophoric molecules, as needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain one or two unique cysteine residues can be constructed for specific labeling with one or two identical fluorescent probes, so-called donors (d). Fluorescence depolarization experiments on double-labeled Cys mutant monitor both reorientational motions of the d molecules, as well as the rate of intramolecular energy migration. In this report a model that accounts for these contributions to the fluorescence anisotropy is presented and experimentally tested. Mutants of a protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1), containing one or two cysteine residues, were labeled with sulfhydryl specific derivatives of 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacence (BODIPY). From the rate of energy migration, the intramolecular distance between the d groups was calculated by using the Forster mechanism and by accounting for the influence of local anisotropic orientation of the d molecules. The calculated intramolecular distances were compared with those obtained from the crystal structure of PAI-1 in its latent form. To test the stability of parameters extracted from experiments, synthetic data were generated and reanalyzed.

Biochemical and biophysical studies of reactive center cleaved plasminogen activator inhibitor type 1. The distance between P3 and P1' determined by donor-donor fluorescence energy transfer.

Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). In accordance with other serpins, PAI-1 is thought to undergo a conformational change upon reactive center cleavage. In this study we have developed methods to produce and purify reactive center cleaved wild-type PAI-1 and characterized this molecular form of PAI-1 by biochemical and biophysical methods. Incubation with Sepharose-bound trypsin caused cleavage only at the P1-P1' bond in the reactive center and resulted in 39- and 4-kDa polypeptides, strongly held together by noncovalent interactions. Circular dichroism measurements suggest that the reactive center cleavage triggers larger conformational changes than the conversion from the active to the latent form. Cleaved PAI-1 did not bind to either PAs or vitronectin but retained the heparin-binding capacity. To study the structure of cleaved PAI-1 by polarized fluorescence spectroscopy and to measure intramolecular distances, we used cysteine substitution mutants to which extrinsic fluorescence probes were attached. These studies revealed increasing orientational freedom of probes in the P3 and P1' positions upon cleavage. Distance measurements based on fluorescence energy transfer between probes in positions P3 and P1' indicate that these residues are separated by at least 68 +/- 10 A in cleaved PAI-1.

Secondary structure of NADPH: protochlorophyllide oxidoreductase examined by circular dichroism and prediction methods.

To study the secondary structure of the enzyme NADPH: protochlorophyllide oxidoreductase (PCOR), a novel method of enzyme isolation was developed. The detergent isotridecyl poly-(ethylene glycol) ether (Genapol X-080) selectively solubilizes the enzyme from a prolamellar-body fraction isolated from wheat (Triticum aestivum L.). The solubilized fraction was further purified by ion-exchange chromatography. The isolated enzyme was studied by fluorescence spectroscopy at 77 K, and by CD spectroscopy. The fluorescence-emission spectra revealed that the binding properties of the substrate and co-substrate were preserved and that photo-reduction occurred. The CD spectra of PCOR were analysed for the relative amounts of the secondary structures, alpha-helix, beta-sheet, turn and random coil. The secondary structure composition was estimated to be 33% alpha-helix, 19% beta-sheet, 20% turn and 28% random coil. These values are in agreement with those predicted by the Predict Heidelberg Deutschland and self-optimized prediction method from alignments methods. The enzyme has some amino acid identity with other NADPH-binding enzymes containing the Rossmann fold. The Rossmann-fold fingerprint motif is localized in the N-terminal region and at the expected positions in the predicted secondary structure. It is suggested that PCOR is anchored to the interfacial region of the membrane by either a beta-sheet or an alpha-helical region containing tryptophan residues. A hydrophobic loop-region could also be involved in membrane anchoring.

Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1: conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin.

Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)

Polarized fluorescence and absorption spectroscopy of 1,32-dihydroxy-dotriacontane-bis-rhodamine 101 ester. A new and lipid bilayer-spanning probe.

We report on the properties of 1,32-dihydroxy-dotriacontane-bis-rhodamine 101 ester (Rh101C32Rh101) in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and in liquid solvents. The results are compared with those of rhodamine 101 octadecanyl ester (Rh101C18). Both molecules are solubilized in the lipid bilayer and the Rh101 moieties are anchored in the lipid-water interface, so that the electronic transition dipole moments (S 0 ↔S 1) are oriented preferentially in the plane of the bilayer. At low concentrations of the dyes in lipid bilayers of DOPC, the fluorescence relaxation is single exponential with a lifetime of τ=4.9±0.2 ns. The relative fluorescence quantum yield of ΦC32/ΦC18 ≈ 0.95 in DOPC vesicles. These results strongly suggest that only a small fraction of the Rh101C32Rh101 molecules are quenched, by, for example, intra- or intermolecular dimers in the ground state at mole fractions of less than 0.1% in the lipid bilayers. For Rh101C32Rh101 in lipid vesicles, the steady-state and time-resolved fluorescence anisotropies are compatible with efficient intramolecular electronic energy transfer. It is concluded that nearly every Rh101C32Rh101 molecule is spanning across the lipid bilayer of DOPC.

Fluorescence studies on plasminogen activator inhibitor 1: reactive centre cysteine mutants remain active after fluorophore attachment.

To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes. After expression in E. coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1). Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1. The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed. The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration. The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion. The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys. No significant spectral changes could be seen when the P3cys mutant was transferred to latency. In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine. Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex.

Phase equilibria and formation of vesicles of dioleoylphosphatidylcholine in glycerol/water mixtures.

The lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) forms a lamellar liquid crystalline phase (L alpha) in arbitrary mixtures of glycerol and water. The phase has been characterized by means of X-ray diffraction, 31P-NMR spectroscopy and differential scanning calorimetry (DSC). In the L alpha state, and for DOPC concentrations greater than 50% (w/w), the thickness of the lipid bilayer decreases, while the area of the polar head group increases with increasing glycerol concentration. The phase transition from gel to L alpha state occurs in the range of 240 to 260 K. Contrary to a previous (McDaniel, R.V., McIntosh, T.J. and Simon, S.A. (1983) Biochim. Biophys. Acta 731, 97) study of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) we find that in the gel state, the thickness of the DOPC lipid bilayer is greater than that in the L alpha state. This suggests that in the gel state, the lipid acyl chains of DOPC are in extended configuration. The lamellar phase reaches its maximum swelling at about 50% (w/w) of DOPC. At lower DOPC concentrations a two-phase system is formed where the lamellar phase exists in equilibrium with excess of solvent. Unilamellar vesicles can be prepared from a diluted suspension of the lamellar phase either by using the sonicator or extruder technique. We show this by means of 31P-NMR, EPR and fluorescence spectroscopy. The mean radius of the vesicles, prepared by a sonicator, has been determined at different glycerol/water mixtures. It is found to decrease continuously from 100 A at 100% water to a minimum of 75 A at about 50% water in the solvent mixture. By further decreasing the water content in the solution, the radius rapidly increases, and a mean radius of 450 A is estimated at a water content of 10%. The rotational relaxation times of a fluorescent probe and two EPR spin probes, solubilized in DOPC vesicles, have been measured at different glycerol/water mixtures. It is found that the rotational rates are always much slower in the systems containing glycerol.

A substrate-like form of plasminogen-activator-inhibitor type 1. Conversions between different forms by sodium dodecyl sulphate.

Recombinant plasminogen-activator-inhibitor type 1 (PAI-1) purified in an active form from Escherichia coli and eucaryotic cells was found to contain a mixture of three functionally distinct forms: an active form that forms complexes with plasminogen activators (PAs), an inactive (latent) form that remains intact after incubation with PAs, and a substrate-like form which is easily cleaved by PAs. Since active PAI-1 purified from bacteria (rpPAI-1) contains only trace amounts of the inactive latent and the substrate-like forms, this material was used to study the effect of sodium dodecyl sulphate (SDS) on the structure and function of active PAI-1. After treatment with 0.01% SDS, active rpPAI-1 was converted to an inactive form that did not form complexes with PAs, but exhibited characteristics similar to those of latent PAI-1. After treatment with 0.1% SDS, PAI-1 lost its inhibitory activity and was cleaved as a substrate in the reactive center. Circular dichroism spectral analysis reveals that SDS changed the conformation of PAI-1 dramatically, mainly by increasing its alpha-helical content.

Sequence specific 1H-NMR assignments and secondary structure of a carboxy-terminal functional fragment of apolipoprotein CII.

The structural properties of a synthetic fragment of human apolipoprotein CII (apoCII) has been studied by circular dichroism and proton nuclear magnetic resonance. The fragment corresponds to the carboxy-terminal 30 amino acid residues and retains the ability of apoCII to activate lipoprotein lipase. Like native apoCII, the fragment has a tendency to self-associate in pure aqueous solution. Addition of 1,1,1,3,3,3-hexafluoro-2-isopropanol to aqueous solvent dissolves the aggregates and leads to an increase in the alpha-helical content of the peptide, probably by stabilizing transient helical structures. The resonances in the 1H-NMR spectrum of the fragment in 35% (CF3)2CHOH were assigned through standard procedures from nuclear Overhauser enhancement spectroscopy, correlated spectroscopy and total correlated spectroscopy experiments. The NMR data indicates the formation of a stable alpha helix spanning Ile66-Gly77. Another alpha helical turn may be formed between Lys55 and Ala59 and possibly span even further towards the carboxyl terminus. These structural elements are different from those previously predicted for this part of the sequence of apoCII.