Oligomerization of hydrophobin SC3 in solution: from soluble state to self-assembly

Hydrophobin SC3 is a protein with special self-association properties that differ depending on whether it is in solution, on an air/water interface or on a solid surface. Its self-association on an air/water interface and solid surface have been extensively characterized. The current study focuses on its self-association in water because this is the starting point for the other two association processes. Size-exclusion chromatography was used to fractionate soluble-state SC3. Real-time multiangular light scattering detection of the eluate indicated that SC3 mainly exists as a dimer in buffer, accompanied with a small amount of monomer, tetramer, and larger aggregates. Dimeric SC3 has very likely an elongated shape, as indicated by the hydrodynamic radius determined by using dynamic light scattering (DLS) and fluorescence anisotropy measurements on dansyl-labeled SC3. Size-exclusion chromatography experiments also indicated that the protein oligomerizes very slowly at low temperature (4 degrees C) but rather rapidly at room temperature. Ionic strength plays an important role in the oligomerization; a short-lived monomeric SC3 species could be observed in pure water. Oligomerization was not affected by low pH but was accelerated by high pH. Fluorescence resonance energy transfer showed that dissociation occurred when the protein concentration was lowered; a large population of oligomers, presumably dimers, dissociate when the protein concentration is <4.5 microg/mL. This value is similar to the critical concentration for SC3 self-assembly. Therefore, dimeric SC3 is indicated to be the building block for both aggregation in solution and self-assembly at hydrophobic/hydrophilic interfaces.     

Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV

We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.     

Voltage sensing by fluorescence resonance energy transfer in single cells.

A new mechanism has been developed for achieving fast ratiometric voltage-sensitive fluorescence changes in single cells using fluorescence resonance energy transfer. The mechanism is based on hydrophobic fluorescent anions that rapidly redistribute from one face of the plasma membrane to the other according to the Nernst equation. A voltage-sensitive fluorescent readout is created by labeling the extracellular surface of the cell with a second fluorophore, here a fluorescently labeled lectin, that can undergo energy transfer with the membrane-bound sensor. Fluorescence resonance energy transfer between the two fluorophores is disrupted when the membrane potential is depolarized, because the anion is pulled to the intracellular surface of the plasma membrane far from the lectin. Bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols, where alkyl is n-hexyl and n-decyl (DiSBA-C6-(3) and DiSBA-C10-(3), respectively) can function as donors to Texas Red labeled wheat germ agglutinin (TR-WGA) and acceptors from fluorescein-labeled lectin (FI-WGA). In voltage-clamped fibroblasts, the translocation of these oxonols is measured as a displacement current with a time constant of approximately 2 ms for 100 mV depolarization at 20 degrees C, which equals the speed of the fluorescence changes. Fluorescence ratio changes of between 4% and 34% were observed for a 100-mV depolarization in fibroblasts, astrocytoma cells, beating cardiac myocytes, and B104 neuroblastoma cells. The large fluorescence changes allow high-speed confocal imaging.     

Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates

Hydrophobins self assemble into amphipathic films at hydrophobic-hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the alpha-helical state that is formed upon binding to a hydrophobic solid, and the beta-sheet state, which is formed at the air-water interface. Here, we show that the formation of the beta-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the alpha-helical state of SC3. The second intermediate is rich in beta-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets.     

Effect of fluorescence quenching model on quinoline derivative with cobalt chloride metal ion using steady state and time resolved spectroscopic methods

Quinoline derivative, i.e. quinilone yellow with the scientific name [sodium 2-(2,3-dihydro-1,3-dioxo-1H-inden-2-yl)quinoline-6,8-disulphonate] (SQDS) is analysed for fluorescence resonance energy transfer (FRET). Fluorescence quenching mechanism is studied by employing steady state and transient state spectroscopic measurements. Cobalt chloride is used as quencher in the present study. Linearity was observed in Stern–Volmer plots for transient state as well as steady state. This was further attributed to a mechanism of collisional quenching. Efficiency in fluorescence quenching is observed as there is a correlation between quenching constants of both transient and steady state. A significant energy transfer is reported between metal ions and SQDS molecule, according to FRET theory. Characterization results are studied and analysed. Application in the field of non-linear optics are predicted for SQDS. With Kurtz and Perry powder technique, SHG (second harmonic generation) efficiency was measured using Q-switched mode locked Nd:YAG laser emitting 1064 nm the first time with this compound.     

Molecular self-assembling and the fluorescence enhancement in morin-Al3+-cetyltrimethylammonium bromide-protein system

Fluorescence enhancement effect of the morin-Al3+-cetyltrimethylammonium bromide (CTAB)-bovine serum albumin (BSA) system is reported here and the interaction mechanism is studied using fluorescence, resonance light scattering (RLS), absorption spectroscopy, Far-UV circular dichroism (CD) spectrum and small angle X-ray scattering (SAXS) measurement. It is considered that protein can bind with Al3+, morin and CTAB through self-assembling function with electrostatic attraction, hydrogen bond, hydrophobic interaction and Vander Waal force etc, and forms a supermolecular association with multilayer structure, in which morin-Al3+ is clamped between BSA and CTAB. In this system, the fluorescence enhancement of morin originates from both intermolecular energy transfer between BSA and morin, and the hydrophobic microenvironment provided by BSA and CTAB. Whereas Al3+ plays a key role for the enhancement of energy transfer efficiency because it provides an efficient channel for the energy transfer between BSA and morin.     

Compact dimension of denatured states of staphylococcal nuclease

Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.     

Fluorescence detected magnetic resonance of the primary donor and inner core antenna chlorophyll in Photosystem I reaction centre protein: Sign inversion and energy transfer

The Photosystem I reaction centre protein CP1, isolated from barley using polyacrylamide gel electrophoresis showed an EPR (Electron Paramgnetic Resonance) spectrum with the polarisation pattern AEEAAE, typical of the primary donor triplet state ³P700, created via radical pair formation and recombination. ³P700 could also be detected by Fluorescence Detected Magnetic Resonance (FDMR) at _f > 700 nm even in the presence of a large number of chlorophyll antennae. Its zero field splitting parameters, D=282.5×10^-4 cm^-1 and E=38.5×10^-4 cm^-1, were independent of the detection wavelength, and agreed with ADMR (Absorption Detected Magnetic Resonance) and EPR values. The signs of the ³P700 D+E and D-E transitions were positive (increase in fluorescence intensity on applying a resonance microwave field). In contrast, in the emission band 685 < _f < 700 nm FDMR spectra with negative D+E and D-E transitions were detected, and the D value was wavelength-dependent. These FDMR results support an excitation energy transfer model for CP1, derived from time-resolved fluorescence studies, in which two chlorophyll antenna forms are distinguished, with fluorescence at 685 < _f < 700 nm (inner core antennae, F690), and _f > 700 nm (low energy antenna sites, F720), in addition to the P700. The FDMR spectrum in F690 emission can be interpreted as that of ³P700, observed via reverse singlet excitation energy transfer and added to the FDMR spectrum of the antenna triplet states generated via intramolecular intersystem crossing. This would indicate that reversible energy transfer between F690 and P700 occurs even at 4.2 K.Abbreviations Chl chlorophyll - CP1 core chlorophyll protein of Photosystem I - EPR electron paramagnetic resonance - F690, F720 chlorophyll forms having fluorescence maximum at 690–695 and 720 nm, respectively - F(A)(O)DMR fluorescence (absorption) (optical) detected magnetic resonance - FF fluorescence fading - ISC intramolecular intersystem crossing - _f fluorescence emission wave-length - LHC I light harvesting chlorophyll a/b protein of Photosystem I - P700 primary donor of Photosystem I - PS I Photosystem I - RC reaction centre - RP radical pair - SDS sodium dodecyl sulphate - ZFS zero field splitting     

Cooperative interactions and a non-native buried Trp in the unfolded state of an SH3 domain

The presence of residual structure in the unfolded state of the N-terminal SH3 domain of Drosophila drk (drkN SH3 domain) has been investigated using far- and near-UV circular dichroism (CD), fluorescence, and NMR spectroscopy. The unfolded (U(exch)) state of the drkN SH3 domain is significantly populated and exists in equilibrium with the folded (F(exch)) state under non-denaturing conditions near physiological pH. Denaturation experiments have been performed on the drkN SH3 domain in order to monitor the change in ellipticity, fluorescence intensity, and chemical shift between the U(exch) state and chemically or thermally denatured states. Differences between the unfolded and chemically or thermally denatured states highlight specific areas of residual structure in the unfolded state that are cooperatively disrupted upon denaturation. Results provide evidence for cooperative interactions in the unfolded state involving residues of the central beta-sheet, particularly the beta4 strand. Denaturation as well as hydrogen-exchange experiments demonstrate a non-native burial of the Trp ring within this "cooperative" core of the unfolded state. These findings support the presence of non-native hydrophobic clusters, organised by Trp rings, within disordered states.     

Estimation of the Compaction of the Denatured State by a Protein Variant Involved in a Reverse Hydrophobic Effect

Fluorescence resonance energy transfer methods have been used to evaluate changes in the dimension of the denatured state for position 73 variants of iso-1-cytochrome c that show a reverse hydrophobic effect [Herrmann et al. (1995)]. The experiments take advantage of the Trp 59/heme donor-acceptor pair in cytochrome c. Two large aliphatic variants, Ile 73 and Leu 73, were compared directly to the wild-type protein (lysine 73). The Leu 73 was an outlier in the original work and serves as an internal control. The data show that the volume of the denatured state is contracted by a small but significant degree, 4-6%, for the Ile 73 variant whereas the Leu 73, which does not conform to the reverse hydrophobic effect, shows no significant compaction. Given that position 73 is beyond Trp 59 in the sequence, the denatured state compaction appears to be a global effect.     

Denatured collapsed states in protein folding: example of apomyoglobin.

Experimental approaches, including circular dichroism, small angle X-ray scattering, steady-state fluorescence, and fluorescence energy transfer, were applied to study the 3D-structure of apomyolgobin in different conformational states. These included the native and molten globules, along with either less ordered conformations induced by the addition of anions or completely unfolded states. The results show that the partially folded forms of apomyoglobin stabilized by KCl and/or Na(2)SO(4) under unfolding conditions (pH 2) exhibit a significant amount of secondary structure (circular dichroism), low packing density of protein molecules (SAXS), and native-like dimensions of the AGH core (fluorescence energy transfer). This finding indicates that a native-like tertiary fold of the polypeptide chain, i.e., the spatial organization of secondary structure elements, most likely emerges prior to the formation of the molten globule state.     

Light Harvesting and State Transitions in Cyanobacteria

Cyanobacteria are oxygenic phototrophic prokaryotes and are considered to be the ancestors of chloroplasts. Their photosynthetic machinery is functionally equivalent in terms of primary photochemistry and photosynthetic electron transport. Fluorescence measurements and other techniques indicate that cyanobacteria, like plants, are capable of redirecting pathways of excitation energy transfer from light harvesting antennae to both photosystems. Cyanobacterial cells can reach two energetically different states, which are defined as “State 1” (obtained after preferential excitation of photosystem I) and “State 2” (preferential excitation of photosystem II). These states can be distinguished by static and time resolved fluorescence techniques. One of the most important conclusions reached so far is that the presence of both photosystems, as well as certain antenna components, are necessary for state transitions to occur. Spectroscopic evidence suggests that changes in the coupling state of the light harvesting antenna complexes (the phycobilisomes) to both photosystems occur during state transitions. The finding that the phycobilisome complexes are highly mobile on the surface of the thylakoid membrane (the mode of interaction with the thylakoid membrane is essentially unknown), has led to the proposal that they are in dynamic equilibrium with both photosystems and regulation of energy transfer is mediated by changes in affinity for either photosystem.     

Interaction of N-methyl-anthraniloyl-labelled porcine apolipoprotein A-I with porcine lecithin: Cholesterol acyltransferase: An energy transfer study

• 1.1. To investigate whether a direct protein-protein interaction between apoA-I and lecithin: cholesterol acyltransferase (LCAT) is necessary for the activation of the enzyme, apoA-I was labelled with N-methylisatoic anhydride at lysine residues. The intermolecular resonance energy transfer from tryptophan residues of LCAT (donor) to N-methyl-anthraniloyl (NMA)-labelled apoA-I (NMA-apoA-I) (acceptor) was used as a sensitive fluorescence method for studying molecular interactions.
• 2.2. In the absence of lipids no fluorescence energy transfer was measurable.
• 3.3. Fluorescence energy transfer occurred from LCAT to NMA-apoA-I in the presence of liposomes with phospholipid/cholesterol ratios ranging from 5:1 to 18:1 and regardless whether only 1 or up to 5 NMA-apoA-I molecules resided at the liposome surface.
• 4.4. This indicates a preferred binding of the enzyme directly to or in spatial proximity to the activator protein NMA-apoA-I even if enough space at the liposome surface is available to allow LCAT binding at a distance, where no energy transfer is measurable.

     

不同解折叠状态鸡心脱血红素细胞色素c分子的表面性质

脱血红素细胞色素ｃ的构象与其跨膜转运能力是密切相关的。鸡心脱血红素细胞色素ｃ具有较强的自发折叠的能力,在一定条件下可以得到不同解折叠状态的鸡心脱血红素细胞色素ｃ。本文利用疏水层析、与疏水探针１,８—ＡＮＳ的结合以及色氨酸与结合的ＡＮＳ之间的荧光共振能量转移,比较了不同解折叠状态的鸡心脱血红素细胞色素ｃ分子的表面性质。结果表明,部分折叠的鸡心脱血红素细胞色素ｃ分子获得了一种高度动态的结构,形成了动态的疏水核心;同时,它也具有较强的凝集倾向。这些性质与鸡心脱血红素细胞色素ｃ分子较强的自发折叠能力是一致的,为进一步分析鸡心脱血红素细胞色素ｃ分子的构象及其在跨膜转运过程中与脂的相互作用奠定了基础。     

Plasmonics in Biology and Plasmon-Controlled Fluorescence

Fluorescence technology is fully entrenched in all aspects of biological research. To a significant extent, future advances in biology and medicine depend on the advances in the capabilities of fluorescence measurements. As examples, the sensitivity of many clinical assays is limited by sample autofluorescence, single-molecule detection is limited by the brightness and photostability of the fluorophores, and the spatial resolution of cellular imaging is limited to about one-half of the wavelength of the incident light. We believe a combination of fluorescence, plasmonics, and nanofabrication can fundamentally change and increase the capabilities of fluorescence technology. Surface plasmons are collective oscillations of free electrons in metallic surfaces and particles. Surface plasmons, without fluorescence, are already in use to a limited extent in biological research. These applications include the use of surface plasmon resonance to measure bioaffinity reactions and the use of metal colloids as light-scattering probes. However, the uses of surface plasmons in biology are not limited to their optical absorption or extinction. We now know that fluorophores in the excited state can create plasmons that radiate into the far field and that fluorophores in the ground state can interact with and be excited by surface plasmons. These reciprocal interactions suggest that the novel optical absorption and scattering properties of metallic nanostructures can be used to control the decay rates, location, and direction of fluorophore emission. We refer to these phenomena as plasmon-controlled fluorescence (PCF). We predict that PCF will result in a new generation of probes and devices. These likely possibilities include ultrabright single-particle probes that do not photobleach, probes for selective multiphoton excitation with decreased light intensities, and distance measurements in biomolecular assemblies in the range from 10 to 200 nm. Additionally, PCF is likely to allow design of structures that enhance emission at specific wavelengths and the creation of new devices that control and transport the energy from excited fluorophores in the form of plasmons, and then convert the plasmons back to light. Finally, it appears possible that the use of PCF will allow construction of wide-field optical microscopy with subwavelength spatial resolution down to 25 nm.     

Human serum albumin. Spectroscopic studies of binding and proximity relationships for fatty acids and bilirubin.

Binding and proximity relationships of hydrophobic ligands on human serum albumin have been studied using absorption, fluorescence, circular dichroism, and electron paramagnetic resonance spectroscopy. The ligands studied were bilirubin, two conjugated linear polyene fatty acids, cis-parinaric acid and cis-eleostearic acid, and three nitroxide derivatives of stearic acid with doxyl groups at positions 5, 10, and 12, respectively. Binding of polyene fatty acids was monitored by absorption peak shifts, induced circular dichroism, enhancement of fluorescence, and energy transfer between albumin's single tryptophanyl residue and the polyene chromophore. Induced circular dichroism studies indicate excitonic ligand-ligand interaction between bound fatty acids. Fluorescence enhancement of cis-parinaric acid was analyzed using a stepwise multiple equilibrium model, and six binding constants in the range 10(8) to 10(6) M-1 were obtained, in agreement with previous measurements for other fatty acids. The temperature dependence of the equilibrium constants indicates that the binding enthalpy is nearly zero. Fluorescence energy transfer was similarly used to quantitate bilirubin binding to albumin. Energy transfer, nitroxide quenching of fluorescence, and electron paramagnetic resonance spectroscopy were used to elucidate binding geometries which support and extend proposed structural models for albumin. It is suggested that the first two fatty acids bind side-by-side in an antiparallel fashion in domain III of human serum albumin.     

Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy

The spatiotemporal localization of neuronal signaling is important for triggering neuronal responses in specific locations at precise times. Fluorescence resonance energy transfer imaging enables measurement of spatiotemporal dynamics of signaling activity in live neurons. Although the usefulness of fluorescence resonance energy transfer is well recognized, there are many difficulties in applying it, particularly when imaging in neuronal micro-compartments in light-scattering brain tissue. Fluorescence resonance energy transfer has been imaged using several techniques including intensity-based methods, fluorescence lifetime imaging and fluorescence anisotropy imaging. These methods have different advantages and disadvantages, and thus are suitable in different applications.     

A PDZ domain-based detection system for enzymatic assays

A time-resolved fluorescence resonance energy transfer (TR-FRET) detection method based on the formation of a PDZ domain.peptide ligand complex has been developed for enzymatic assays as an alternative to immuno-based detection strategies. The enzyme substrate is a "masked" biotinylated PDZ domain peptide ligand containing the consensus sequence Ser-X-Val-COOH. The critical residues in the binding consensus sequence of the ligand have been modified, for example, by phosphorylation of Ser or C-terminal extensions, providing binding-incompetent PDZ domain peptides. On processing by the corresponding enzyme, the binding epitope is exposed, and the product sequence is recognized specifically by Eu(3+) chelate-labeled GST-PDZ ([Eu(3+)]GST-PDZ) (GST-PDZ-glutathione S-transferase fused to PDZ domain). A ternary complex is subsequently formed by addition of allophycocyanin-labeled streptavidin ([XL665]SA), which binds to the biotinylated N terminus of the peptide, and detected by TR-FRET. Reported here are examples of the applicability of this detection strategy to three enzymatic systems, an endoprotease, an exoprotease, and a Ser/Thr phosphatase.     

Exponential decay kinetics in "downhill" protein folding

The observation of single-exponential kinetic phases in early stages of protein folding is often interpreted as evidence that these phases are rate limited by significant energy or entropy barriers. However, although the existence of large barriers reliably implies exponential kinetics, the reverse is not necessarily true. A simple model for the hydrophobic collapse of a chain molecule demonstrates that a barrierless or "downhill" diffusional relaxation can give rise to kinetics that are practically indistinguishable from a pure exponential. Within this model, even a highly nonlinear experimental probe such as resonance energy transfer (F?rster transfer) could exhibit a large amplitude decay (greater than 90% in fluorescence) that deviates from a simple exponential by less than 0.5%. Only a detailed analysis of the dynamics is likely to reveal that a free energy barrier is absent.     

Probing the self-assembly and the accompanying structural changes of hydrophobin SC3 on a hydrophobic surface by mass spectrometry

The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate alpha-helical state to the stable end form called the beta-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the alpha-helical state. The beta-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39-S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of alpha-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the beta-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly.