Molecular properties of R-phycocyanin subunits from Polysiphonia urceolata in potassium phosphate buffer.

The two subunits of R-phycocyanin from Polysiphonia urceolata were isolated and renatured. The renatured subunits were characterized by electrophoresis, molecular weights and spectra. The blue-shifted spectra, fluorescence recovery and restoring of the energy transfer suggested correct refolding of the subunits. The molecular properties of the subunits in potassium phosphate buffer (KPB) were investigated in detail. The total fluorescence yields (Q(T)) of the beta subunit declined while the energy transfer efficiency (E(T)) in the beta subunit was promoted with the increase of KPB concentration. On the other hand, both Q(T) and E(T) were enhanced with the increasing of the subunit concentrations. Based on the structural information, the fluorescence quenching in high concentrations of KPB was ascribed to less rigid chromophores caused by the weakening of the hydrogen-bond interaction network, while the enhancement of the fluorescence and E(T) was due to the aggregation of the subunits in the ionic solvent. Aggregation was confirmed by cysteine-assisted promotion of renaturation yield and stability, as well as equilibrium unfolding tests. Optimal conditions were proposed for the refolding/unfolding studies, under which the subunits were mainly monomeric. Compared to that in C-PC, the blue-shifted spectrum of PCB in R-PC is suggested to bring larger energy transfer efficiency, probably due to the necessity of the light harvesting for P. urceolata living in deep water.     

The guanidine like effects of arginine on aminoacylase and salt-induced molten globule state

Aminoacylase is a dimeric enzyme containing one Zn(2+) ion per subunit. The arginine (Arg)-induced unfolding of Holo-aminoacylase and Apo-aminoacylase has been studied by measurement of enzyme activity, fluorescence emission spectra and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra. Besides being the most alkaline amino acid, the arginine molecule contains a positively charged guanidine group, similar to guanidine hydrochloride, and has been used in many refolding systems to suppress protein aggregation. Our results showed that arginine caused the inactivation and unfolding of aminoacylase, with no aggregation during denaturation. A comparison between the unfolding of aminoacylase in aqueous and HCl (pH 7.5) arginine solutions indicated that the guanidine group of arginine had protein-denaturing effects similar to those of guanidine hydrochloride, which might help us understand the mechanism by which arginine suppresses incorrect refolding. The results showed that arginine-denatured aminoacylase could be reactivated and refolded correctly, indicating that arginine is as good a denaturant as the guanidine or urea for study of protein unfolding and refolding. Both the intrinsic fluorescence and the ANS fluorescence spectra showed that the arginine-unfolded aminoacylase formed a molten globule state in the presence of KCl, suggesting that intermediates exist during aminoacylase refolding. The results for the Apo-aminoacylase followed were similar to those for the Holo-enzyme, suggesting that Holo- and Apo-aminoacylase might have a similar unfolding and refolding pathway.     

Equilibrium and kinetic studies on folding of canine milk lysozyme

Thermal and chemical unfolding studies of the calcium-binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped-flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo-canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s(-1). Furthermore, the results show that, unlike its drastic effect on the stability, Ca(2+)-binding only marginally affects the refolding kinetics. During the refolding process of apo-CL non-native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non-native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.     

Nativelike intermediate on the unfolding pathway of pig kidney fructose-1,6-bisphosphatase

The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving as the first intermediate a catalytically inactive tetramer. The evidence that indicates the existence of this intermediate is as follows: (1) the loss of enzymatic activity and the concomitant increase of ANS binding, at low concentrations of Gdn.HCl (midpoint at 0.75 M), are both protein concentration independent, and (2) the enzyme remains in a tetrameric state at 0.9 M Gdn.HCl as shown by size-exclusion chromatography. At slightly higher Gdn.HCl concentrations the inactive tetramer dissociates to a compact dimer which is prone to aggregate. Further evidence for dissociation of tetramers to dimers and of dimers to monomers comes from the concentration dependence of AEDANS-labeled enzyme anisotropy data. Above 2.3 M Gdn.HCl the change of AEDANS anisotropy is concentration independent, indicative of monomer unfolding, which also is detected by a red shift of MIANS-labeled enzyme emission. At Gdn.HCl concentrations higher than 3.0 M, the protein elutes from the size-exclusion column as a single peak, with a retention volume smaller than that of the native protein, corresponding to the completely unfolded monomer. In the presence of its cofactor Mg(2+), the denaturated enzyme could be successfully reconstituted into the active enzyme with a yield of approximately 70-90%. Refolding kinetic data indicate that rapid refolding and reassociation of the monomers into a nativelike tetramer and reactivation of the tetramer are sequential events, the latter involving slow and small conformational rearrangements in the refolded enzyme.     

Deconvolution of C-phycocyanin beta-84 and beta-155 chromophore absorption and fluorescence spectra of cyanobacterium Mastigocladus laminosus.

Absorption and fluorescence spectra of the C-phycocyanin beta-subunit were quantitatively deconvoluted into component spectra of the beta-84 and beta-155 chromophores. The deconvolution procedure was based on a theoretical treatment of polarization properties. Four kinds of spectra (absorption, emission, emission polarization, and excitation polarization) measured on C-phycocyanin isolated from the cyanobacterium Mastigocladus laminosus were used as the experimental data set. Without any assumption of spectral shape, the absorption and fluorescence spectra of both chromophores were unambiguously resolved and their fluorescence quantum yields were evaluated. By combining the spectra of the alpha-subunit, independently measured, with the resolved spectra of the beta-subunit, the fluorescence and fluorescence polarization spectra and the fluorescence quantum yield of the monomer were estimated; they agree with experimental values to within an acceptable error. Further, the matrix of energy transfer rates in the monomer was estimated; it gave a significantly different result (by up to 40%) from previously estimated ones.     

Refolding intermediate of guanidine hydrochloride denatured aminoacylase

The refolding of aminoacylase denatured in 6M guanidine hydrochloride (GdnHCl) has been studied by measuring enzyme activity, fluorescence emission spectra, ANS fluorescence spectra and far-UV circular dichroism spectra. The results showed that GdnHCl-denatured aminoacylase could be refolded and reactivated by dilution. A refolding intermediate was observed for low concentrations of GdnHCl (between 0.5 and 1.2M). This refolding intermediate was characterized by an increased fluorescence emission intensity, a blue-shifted emission maximum, and by increased binding of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). The secondary structure of the intermediate was similar to that of the native enzyme, and was therefore quite similar to the molten globule state often found in the protein folding pathway. Combined with the previous evidence of existence of an intermediate during unfolding process, we therefore proposed that the unfolding and refolding of aminoacylase might share the same pathway. A comparison of the Apo-enzyme and Holo-enzyme showed that there was little effect of the zinc ion on the refolding of the aminoacylase. Our study, the first successful report of the refolding of this metalloenzyme, also showed that lowering the concentration and the temperature of the enzyme improved the refolding rate of aminoacylase. The system therefore provides a useful model to study the refolding of proteins with prosthetic groups.     

Tracking unfolding and refolding of single GFPmut2 molecules

The unfolding and refolding kinetics of >600 single GFPmut2 molecules, entrapped in wet nanoporous silica gels, were followed by monitoring simultaneously the fluorescence emission of the anionic and neutral state of the chromophore, primed by two-photon excitation. The rate of unfolding, induced by guanidinium chloride, was determined by counting the number of single molecules that disappear in fluorescence images, under conditions that do not cause bleaching or photoinduced conversion between chromophore protonation states. The unfolding rate is of the order of 0.01 min(-1), and its dependence on denaturant concentration is very similar to that previously reported for high protein load gels. Upon rinsing the gels with denaturant-free buffer, the GFPmut2 molecules refold with rates >10 min(-1), with an apparently random distribution between neutral and anionic states, that can be very different from the preunfolding equilibrium. A subsequent very slow (lifetime of approximately 70 min) relaxation leads to the equilibrium distribution of the protonation states. This mechanism, involving one or more native-like refolding intermediates, is likely rate limited by conformational rearrangements that are undetectable in circular dichroism experiments. Several unfolding/refolding cycles can be followed on the same molecules, indicating full reversibility of the process and, noticeably, a bias of denaturated molecules toward refolding in the original protonation state.     

金黄色葡萄球菌核酸酶及其类似物盐酸胍变性的活力与构象变化的比较

金黄色葡萄球菌核酸酶（ＳｔａｐｈｌｏｃｏｃｃａｌＮｕｃｌｅａｓｅ,ＳＮａｓｅ）与其类似物（ＳＮａｓｅＲ）的平衡态盐酸胍变性与复性曲线及比活力值均无明显差别。两者变性与复性的构象变化是可逆的,但活力恢复滞后于失活。低浓度盐酸胍对ＳＮａｓｅＲ有２０％左右的激活,而低浓度（０．１２５ｍｏｌ／Ｌ）的ＮａＣｌ可使ＳＮａｓｅＲ的活力提高近一倍。ＳＮａｓｅＲ盐酸胍变性的失活先于构象变化。比较加入底物竞争抑制剂脱氧胸腺嘧啶核苷３＇－５＇－二磷酸（ｐｄＴｐ）后得到的（ＳＮａｓｅＲ＋ｐｄＴｐ＋Ｃａ２＋）三元络合物平衡态盐酸胍变性的Ｔｒｐ与Ｔｙｒ内源荧光的变化,观测到该酶的活性部位先于整体构象发生变化,结果导致ｐｄＴｐ解离常数Ｋｄ增大．     

金黄色葡萄球菌核酸酶及其类似物盐酸胍变性的活力与构象变化的比较

金黄色葡萄球菌核酸酶（ＳｔａｐｈｌｏｃｏｃｃａｌＮｕｃｌｅａｓｅ,ＳＮａｓｅ）与其类似物（ＳＮａｓｅＲ）的平衡态盐酸胍变性与复性曲线及比活力值均无明显差别。两者变性与复性的构象变化是可逆的,但活力恢复滞后于失活。低浓度盐酸胍对ＳＮａｓｅＲ有２０％左右的激活,而低浓度（０．１２５ｍｏｌ／Ｌ）的ＮａＣｌ可使ＳＮａｓｅＲ的活力提高近一倍。ＳＮａｓｅＲ盐酸胍变性的失活先于构象变化。比较加入底物竞争抑制剂脱氧胸腺嘧啶核苷３＇－５＇－二磷酸（ｐｄＴｐ）后得到的（ＳＮａｓｅＲ＋ｐｄＴｐ＋Ｃａ２＋）三元络合物平衡态盐酸胍变性的Ｔｒｐ与Ｔｙｒ内源荧光的变化,观测到该酶的活性部位先于整体构象发生变化,结果导致ｐｄＴｐ解离常数Ｋｄ增大．     

Folding kinetics of the all-beta-sheet protein human basic fibroblast growth factor, a structural homolog of interleukin-1beta

The refolding and unfolding kinetics of the all-beta-sheet protein human basic fibroblast growth factor (hFGF-2) were studied by fluorescence spectroscopy. The kinetics of the unfolding transition are monophasic. The refolding reaction at high and low guanidinium chloride (GdmCl) concentrations is best described by mono- and biphasic folding, respectively. Refolding and unfolding of hFGF-2 (155 amino acids) is very slow compared with other non-disulfide-bonded monomeric proteins of similar size. For example, the rate constant for unfolding at 4.5 mol.liter(-1) GdmCl is 0.006 s(-1), and the refolding rate constants at 0.4 mol.liter(-1) GdmCl are 0.01 s(-1) and 0.0009 s(-1) (15 degrees C, pH 7.0). A characterization of the thermodynamic nature of the folding process using transition state theory revealed that the slow refolding is almost exclusively controlled by entropic factors, namely the strong loss of conformational freedom during refolding. The rate of the slow unfolding kinetics is mainly (and at low denaturant concentrations exclusively) controlled by the large positive change in enthalpy. hFGF-2 shows similar slow folding kinetics to that of its structural homolog interleukin-1beta. Since both proteins show very little sequence identity, it is suggested that their slow folding kinetics are determined by the complex beta-sheet arrangement of the native molecules.     

Fluorescence energy transfer indicates similar transient and equilibrium intermediates in staphylococcal nuclease folding

Fluorescence resonance energy transfer (FRET) is one of the few methods available to measure the rate at which a folding protein collapses. Using staphylococcal nuclease in which a cysteine residue was engineered in place of Lys64, permitted FRET measurements of the distance between the donor tryptophan 140 and 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid-labeled Cys64. These measurements were undertaken on both equilibrium partially folded intermediates at low pH (A states), as well as transient intermediates during stopped-flow refolding. The results indicate that there is an initial collapse of the protein in the deadtime of the stopped-flow instrument, corresponding to a regain of approximately 60% of the native signal, followed by three slower transients. This is in contrast to circular dichroism measurements which show only 20-25% regain of the native secondary structure in the burst phase. Thus hydrophobic collapse precedes the formation of substantial secondary structure. The first two detected transient intermediate species have FRET properties essentially identical with those of the previously characterized equilibrium A state intermediates, suggesting similar structures between the equilibrium and transient intermediates.The effects of anions on the folding of acid-unfolded staphylococcal nuclease, and urea on the unfolding of the resulting A states, indicates that in folding the protein becomes compact prior to formation of major secondary structure, whereas in unfolding the protein expands prior to major loss of secondary structure. Comparison of the kinetics of refolding of staphylococcal nuclease, monitored by FRET, and for a proline-free variant, indicate that folding occurs via two partially folded intermediates leading to a native-like species with one (or more) proline residues in a non-native conformation. For the A states an excellent correlation between compactness measured by FRET, and compactness determined from small-angle X-ray scattering, was observed. Further, a linear relationship between compactness and free energy of unfolding was noted. Formation of soluble aggregates of the A states led to dramatic enhancement of the FRET, consistent with intermolecular fluorescence energy transfer.     

Folding of Escherichia coli DsbC: characterization of a monomeric folding intermediate

The homodimeric protein DsbC is a disulfide isomerase and a chaperone located in the periplasm of Escherichia coli. We have studied the guanidine hydrochloride (GdnHCl)-induced unfolding and refolding of DsbC using mutagenesis, intrinsic fluorescence, circular dichroism spectra, size-exclusion chromatography, and sedimentation velocity analysis. The equilibrium refolding and unfolding of DsbC was thermodynamically reversible. The equilibrium folding profile measured by fluorescence excited at 280 nm exhibited a three-state transition profile with a stable folding intermediate formed at 0-2.0 M GdnHCl followed by a second transition at higher GdnHCl concentrations. Sedimentation velocity data revealed dissociation of the dimer to the monomer over the concentration range of the first transition (0-2.0 M). In contrast, fluorescence emission data for DsbC excited at 295 nm showed a single two-state transition. Fluorescence emission data for the equilibrium unfolding of the monomeric G49R mutant, excited at either 295 or 280 nm, indicated a single two-state transition. Data obtained for the dimeric Y52W mutant indicated a strong protein concentration dependence of the first transition but no dependence of the second transition in equilibrium unfolding. This suggests that the fluorescence of Y52W sensitively reports conformational changes caused by dissociation of the dimer. Thus, the folding of DsbC follows a three-state transition model with a monomeric folding intermediate formed in 0-2.0 M GdnHCl. The folding of DsbC in the presence of DTT indicates an important role for the non-active site disulfide bond in stabilizing the conformation of the molecule. Dimerization ensures the performance of chaperone and isomerase functions of DsbC.     

Metal ion-induced stabilization and refolding of anticoagulation factor II from the venom of Agkistrodon acutus

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF II, holo-ACF II, and Tb(3+)-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism (CD). Metal ions were found to increase the structural stability of ACF II against GdnHCl and irreversible thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF II and Tb(3+)-ACF II is a two-state process with no detectable intermediate state, while the GdnHCl-induced unfolding/refolding of holo-ACF II in the presence of 1 mM Ca(2+) follows a three-state transition with an intermediate state. Ca(2+) ions play an important role in the stabilization of both native and I states of holo-ACF II. The decalcification of holo-ACF II shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, while the reconstitution of apo-ACF II with Tb(3+) ions shifts the initial zone of the denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.1 M GdnHCl) at which refolding from the fully denatured state of apo-ACF II to the I state of holo-ACF II or to the native state of Tb(3+)-ACF II can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF II, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ion-induced refolding provide evidence for the fact that the first phase of Tb(3+)-induced refolding should involve the formation of the compact metal-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

Ca(II)- and Tb(III)-induced stabilization and refolding of anticoagulation factor I from the venom of Agkistrodon acutus

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF I, holo-ACF I, and Tb(3+)-reconstituted ACF I in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism. Metal ions were found to increase the structural stability of ACF I against GdnHCl and thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF I and Tb(3+)-ACF I is a two-state process with no detectable intermediate state(s), whereas the GdnHCl-induced unfolding/refolding of holo-ACF I in the presence of 1 mM Ca(2+) follows a three-step transition, with intermediate state a (Ia) and intermediate state b (Ib). Ca(2+) ions play an important role in the stabilization of the Ia and Ib states. The decalcification of holo-ACF I shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, whereas the reconstitution of apo-ACF I with Tb(3+) ions shifts the initial zone of denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.0 M GdnHCl) at which refolding from the fully denatured state of apo-ACF I to the Ib state of holo-ACF I or to the native state of Tb(3+)-ACF I can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF I, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ions-induced refolding provide evidence that the compact Tb(3+)-binding region forms first, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

Steady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane

We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.     

High-sensitivity fluorescence anisotropy detection of protein-folding events: application to alpha-lactalbumin

An experimental procedure has been devised to record simultaneously fluorescence intensity and fluorescence anisotropy. A photoelastic modulator on the excitation beam enables the anisotropy signal to be recorded in one pass using a single photomultiplier tube and eliminates the need for a polarizer on the emission path. In conjunction with a stopped-flow mixer, providing a time-resolved capability, this procedure was used to study the refolding of apo alpha-lactalbumin following dilution from guanidinium chloride. Although the fluorescence intensity does not change detectably, the fluorescence anisotropy was found to resolve the conformational changes occurring between the initial unfolded state and the molten globule state formed either kinetically during refolding at pH 7.0 or at equilibrium at pH 2.0 (A-state). This result provides further evidence that fluorescence anisotropy is a valuable probe of protein structural transitions and that the information it provides concerning the rotational mobility of a fluorophore can be complementary to the information about the local environment provided by fluorescence intensity.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: equilibrium unfolding studies

UDP-galactose 4-epimerase from yeast (Kluyveromyces fragilis) is a homodimer of total molecular mass 150 kDa having possibly one mole of NAD/dimer acting as a cofactor. The molecule could be dissociated and denatured by 8 M urea at pH 7.0 and could be functionally reconstituted after dilution with buffer having extraneous NAD. The unfolded and refolded equilibrium intermediates of the enzyme between 0-8 M urea have been characterized in terms of catalytic activity, NADH like characteristic coenzyme fluorescence, interaction with extrinsic fluorescence probe 1-anilino 8-naphthelene sulphonic acid (ANS), far UV circular dichroism spectra, fluorescence emission spectra of aromatic residues and subunit dissociation. While denaturation monitored by parameters associated with active site region e.g. inactivation and coenzyme fluorescence, were found to be cooperative having delta G between -8.8 to -4.4 kcals/mole, the overall denaturation process in terms of secondary and tertiary structure was however continuous without having a transition point. At 3 M urea a stable dimeric apoenzyme was formed having 65% of native secondary structure which was dissociated to monomer at 6 M urea with 12% of the said structure. The unfolding and refolding pathways involved identical structures except near the final stage of refolding where catalytic activity reappeared.     

Aspartate-induced aminoacylase folding and forming of molten globule

Aspartate is an osmolyte found in some marine invertebrates and cyclostome fish. The aspartate-induced unfolding of N-acylamino acid amido hydrolase (aminoacylase) has been studied by measuring enzyme activity, fluorescence emission spectra, 8-anilino-1-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that aspartate caused the inactivation and unfolding of aminoacylase. Surprisingly, increasing concentration of aspartate showed the "acid-induced folding", which used to be seen only in strong acids or salts at much lower pH. Although aspartate has the pI of 2.77 that is the lowest among all the free amino acids, it is actually a weak acid. It is thus of great interest why it causes this phenomenon to happen. The relative change of intrinsic fluorescence and ANS binding spectra have shown that there existed a stable molten globule state of aminoacylase with slightly disrupted tertiary structure and more hydrophobic surface. The molten globule state indicates that intermediates existed during aminoacylase refolding process. Unlike the other acids, such as trichloroacetic acid, there is no precipitation observed as the aspartate concentrations increased. It suggests the aspartate anions have an osmotic effect for the molten globule formed during unfolding process. Binding of aspartate anion to the protonated protein, which minimizes the intramolecular repulsion, might explain the osmotic effect of this amino acid in the nature. The results also showed the Apo-aminoacylase followed similar rules as Holo-enzyme, which suggested the zinc ion may play more important roles on activity other than structure.     

Confocal microscopic dual-laser dual-polarization FRET (2polFRET) at the acceptor side for correlating rotations at different distances on the cell surface

Relationship of donor and acceptor fluorescence anisotropies as well as efficiency of fluorescence resonance energy transfer (FRET) has been investigated in a confocal microscope in the context of FRET systems comprised of donor and acceptor-labeled MHCI and MHCII receptors on the surface of Kit-225 K6 human T-cells. The measurements have been carried out in a 2-laser, 5-signal platform where the total donor fluorescence intensity and 2 acceptor fluorescence intensities with their anisotropies – one at the donor's excitation wavelength, the other at the acceptor's excitation wavelength – have been detected. This configuration enabled the determination of FRET efficiency and correlating it with the two acceptor fluorescence anisotropies as a kind of calibration. Estimations for the FRET-enhanced donor fluorescence anisotropy, the directly excited acceptor fluorescence anisotropy, and the fluorescence anisotropy of sensitized emission have been obtained. Procedures for determining FRET by measuring only the total donor intensity and the acceptor intensity and its anisotropy, or two acceptor intensities and their anisotropies have been elaborated, the errors of which have been estimated based on the fluorescence anisotropy values obtained in the calibration with the method of flow cytometric energy transfer (FCET).The combined detection of the donor and acceptor fluorescence anisotropies enabled also the determination of the lower and upper limits of the orientation factor for FRET (κ²). An increase in range for κ² with increasing FRET efficiency has been observed, with average κ² values different from the dynamic random average of 2/3. These observations call for the need of κ² determination in proximity measurements, where the donor and acceptor orientations are not predictable.An increasing range of κ² with increasing intermolecular proximity of the MHCI and MHCII receptors has been observed. This indicates that molecular flexibility in the clusters of the MHCI and MHCII receptors reduces with increasing cluster density, i.e. a “fluidity gradient” exists in the clusters. More specifically, the local density dependent flexibility can also be taken as a direct proof for that the association of these receptors is non-random, but mediated by some type of physical interaction, a finding as a benefit of FRET detection by polarization spectroscopy.Two new quantities – the quenched donor fluorescence anisotropy and a fluorescence anisotropy analogue, the “dissymmetry index” of the polarized FRET efficiency components – have also been introduced for the characterization of the orientational dynamics of the excited state during FRET.     

Structural changes of yellow Cameleon domains observed by quantitative FRET analysis and polarized fluorescence correlation spectroscopy

Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding.