Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy

The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct processes that occur in different GdmCl concentration ranges. From 0 to 0.5 M GdmCl, a slight alteration of the tubulin heterodimer occurs, as evidenced by a small, but reproducible increase in the rotational correlation time of the protein and a sharp decrease in the secondary structure monitored by CD. In the range 0.5-1.5 M GdmCl, significant decreases in the steady-state anisotropy and average lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl range, the number of protein molecules (labeled with Alexa 488), as determined by two-photon FCS measurements, increases by a factor of two, indicating dissociation of the tubulin dimer into monomers. From 1.5 to 4 M GdmCl, these monomers unfold, as evidenced by the continual decrease in the tryptophan steady-state anisotropy, average lifetime, and rotational correlation time, concomitant with secondary structural changes. These results help to elucidate the unfolding pathway of the tubulin heterodimer and demonstrate the value of FCS measurements in studies on oligomeric protein systems.     

Combining time‐resolved fluorescence with synchronous fluorescence spectroscopy to study bovine serum albumin‐curcumin complex during unfolding and refolding processes

We investigated the complex interaction between bovine serum albumin (BSA) and curcumin by combining time‐resolved fluorescence and synchronous fluorescence spectroscopy. The interaction was significant and sensitive to fluorescence lifetime and synchronous fluorescence characteristics. Binding of curcumin significantly shortened the fluorescence lifetime of BSA with a bi‐molecular quenching rate constant of k_q = 3.17 × 10¹² M^‐1s^‐1. Denaturation by urea unfolded the protein molecule by quenching the fluorescence lifetime of BSA. The tyrosine synchronous fluorescence spectra were blue shifted whereas the position of tryptophan synchronous fluorescence spectra was red shifted during the unfolding process. However, denaturation of urea had little effect on the synchronous fluorescence peak of tyrosine in curcumin‐BSA complex except in the low concentration range; however, it shifted the peak to the red, indicating that curcumin shifted tryptophan moiety to a more polar environment in the unfolded state. Decreases in the time‐resolved fluorescence lifetime and curcumin‐BSA complex during unfolding were recovered during refolding of BSA by a dilution process, suggesting partial reversibility of the unfolding process for both BSA and curcumin‐BSA complex. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorescence decay of sarcoplasmic reticulum ATPase. Ligand binding and hydration effects

Multifrequency phase-modulation lifetime data were acquired for sarcoplasmic reticulum Ca2+-ATPase. The intrinsic tryptophan fluorescence decay was complex and was fitted either with three exponentials or with bimodal Lorentzian distributions of lifetimes. Ca2+ binding to the high affinity sites in the ATPase produced an increase of 11% in the center of the main component of the bimodal distribution, shifting the lifetime from 4.04 to 4.50 ns. The effects of solvent on the ATPase were studied with the enzyme dissolved in reverse micelles of detergent bis-(2-ethylhexyl)sulfosuccinate in hexane. Increasing amounts of water up to a water/bis-(2-ethylhexyl)sulfosuccinate molar ratio of 4 produced marked changes in the fluorescence emission of the protein. Comparison of data obtained for micellar solutions of tryptophan or ATPase indicated that the tryptophan residues in the protein are protected from exposure to water. Correlation of water effects on emission intensity and lifetimes suggested that interaction with solvent may result in structural changes that cause a mixture of dynamic and static quenching of ATPase intrinsic fluorescence. Evidence for an effect of hydration on the structure of the active site was obtained by measurements of the fluorescence properties of fluorescein isothiocianate-labeled ATPase in reverse micelles.     

Intrinsic Fluorescence in Endoglucanase and Cellobiohydrolase from Trichoderma pseudokiningii S-38: Effects of pH, Quenching Agents, and Ligand Binding

To gain further insight into the difference in substrate specificity between endoglucanase and cellobiohydrolase, the intrinsic fluorescence properties of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from Trichoderma pseudokiningii S-38 were investigated. The results for the spectral characteristics, ligand binding and fluorescence quenching suggest that the fluorescence of two enzymes comes from tryptophan residues, and that tryptophan residue(s) may be involved in the function of the two enzymes. The results also suggest that the binding tryptophan in EG I may be more exposed to solvent than that in CBH I. This interpretation is supported by the observations that the effects of pH upon the fluorescence of EG I are greater than that of CBH I; spectral shifts are different in EG I and CBH I under various conditions, and fluorescence lifetime changes caused by cellobiose binding are larger for EG I than for CBH I.     

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.     

Evidence of conformational switch in Streptococcus pneumoniae FtsZ during polymerization

FtsZ, the master coordinator of bacterial cell division, assembles into filaments in the presence of nucleotide. FtsZ from Streptococcus pneumoniae bears two tryptophan residues (W294 and W378) in its amino acid sequence. The tryptophan fluorescence of FtsZ increases during the assembly of FtsZ. We hypothesized that this increase in the fluorescence intensity was due to the change in the environment of one or both tryptophan residues. To examine this, we constructed two mutants (W294F and W378F) of FtsZ by individually replacing tryptophan with phenylalanine. The mutants displayed similar secondary structures, GTPase activity, and polymerization ability as the wild type FtsZ. During the polymerization, only one tryptophan (W294) showed an increase in its fluorescence intensity. Using time‐correlated single‐photon counting, the fluorescence lifetime of W294 was found to be significantly higher than W378, indicating that W294 was more buried in the structure than W378. The lifetime of W294 further increased during polymer formation, while that of W378 remained unchanged. Fluorescence quenching experiment suggested that the solvent exposure of W294 reduced during the polymerization of FtsZ. W294 is located near the T‐7 loop of the protein, a region important for the monomer‐monomer interaction during the formation of a protofilament. The results indicated that the region around W294 of S. pneumoniae FtsZ undergoes a conformational switch during polymerization as seen for FtsZ from other bacteria.     

Fluorescence spectral resolution of myelin basic protein conformers in complexes with lysophosphatidylcholine

The structure of (Deibler) myelin basic protein in solution and in a lysolecithin lipid complex has been studied by using the emission properties of the single tryptophan residue of the protein (Trp-115). The studies have been carried out using both static and time-resolved fluorescence techniques. Relative to the free protein, the lipid bound myelin basic protein showed a, twofold increase in fluorescence intensity and a marked blue-shift in the emission maximum wavelength. The multiexponential fluorescence decays and the decay associated spectra indicated that the protein exists in at least three different conformations both in buffer and in lipids. Fluorescence polarization and acrylamide quenching experiments showed that the tryptophan containing region of the protein is embedded in the lipid matrix. The binding of the protein to the lipid appears to be comparable with that predicted for the interaction of amphipathic helices with nonpolar lipids.     

Measurements of label free protein concentration and conformational changes using a microfluidic UV-LED method

This paper presents a microchip-based system for measuring concentrations and dynamic conformational changes in proteins without any use of extrinsic fluorescent labeling. The microchannel flow of protein molecules was integrated with an ultraviolet light-emitting diode (UV-LED, lambda ex = 295 nm) and a photodetector (lambda em = 330 nm). The intrinsic fluorescence shift, arising from selectively exciting aromatic amino acid tryptophan (Trp), was monitored to quantify refolding pathways by dynamically varying the concentration of the chemical denaturant, urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls, rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. The concentrations of proteins were first measured using the UV-LED microfluidic platform, and the data showed detection limits down to 72, 128, and 250 nM for tryptophan, bovine serum albumin (BSA), and bovine carbonic anhydrase (BCA), respectively. To validate the protein assay method, folding transition experiments were performed using a well-characterized protein, BSA. The microchip protein refolding transitions using intrinsic fluorescence were well-correlated with conventional fluorometer experiments. The microfluidic platform facilitates refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material. The technique offers a real alternative to bulky microfluidic systems consisting of large and expensive laser-based designs.     

Membrane interaction and activity of the glycolipid transfer protein

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.     

The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions

Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady‐state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high‐throughput drug screening campaigns.     

Binding of Janus kinase inhibitor tofacitinib with human serum albumin: multi-technique approach

In this report, we have investigated the binding affinity of tofacitinib with human serum albumin (HSA) under simulated physiological conditions by using UV–visible spectroscopy, fluorescence quenching measurements, dynamic light scattering (DLS), differential scanning calorimetry (DSC) and molecular docking methods. The obtained results demonstrate that fluorescence intensity of HSA gets quenched by tofacitinib and quenching occurs in static manner. Binding parameters calculated from modified Stern–Volmer equation shows that the drug binds to HSA with a binding constant in the order of 10⁵. Synchronous fluorescence data deciphered the change in the microenvironment of tryptophan residue in HSA. UV spectroscopy and DLS measurements deciphered complex formation and reduction in hydrodynamic radii of the protein, respectively. Further DSC results show that tofacitinib increases the thermo stability of HSA. Hydrogen bonding and hydrophobic interaction are the main binding forces between HSA and tofacitinib as revealed by docking results.     

Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants.

Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389-396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683-6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741-750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS)     

渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦(Triticum sativum L.)根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯(DPH)荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H     

Interaction between protein kinase C and sphingomyelin/cholesterol

Physical characteristics of binding of protein kinase C with sphingomyelin/cholesterol lipid bilayers were analysed using three complementary approaches: acrylodan fluorescence, fluorescence energy transfer and quenching of tryptophan fluorescence. It was demonstrated that sphingomyelin/cholesterol lipid membranes were available for protein kinase C binding. The intensity of the binding was dependent on the sphingomyelin content. The results of quenching of intrinsic tryptophan fluorescence showed that the enzyme molecule penetrated the sphingomyelin/cholesterol lipid bilayer to the C-16 position of labeled fatty acid probes. Our results also showed sphingomyelin itself restrains protein kinase C activity. A possible explanation for our results is that caveolae function as signaling storage devices.     

Interaction with membrane mimics of transmembrane fragments 16 and 17 from the human multidrug resistance ABC transporter 1 (hMRP1/ABCC1) and two of their tryptophan variants

The human multidrug resistance-associated protein 1 (hMRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily. Together with P-glycoprotein (ABCB1) and the breast cancer resistance protein (BCRP/ABCG2), hMRP1 confers resistance to a large number of structurally diverse drugs. The current topological model of hMRP1 includes two cytosolic nucleotide-binding domains and 17 putative transmembrane (TM) helices forming three membrane-spanning domains. Mutagenesis and labeling studies have shown TM16 and TM17 to be important for function. We characterized the insertion of the TM16 fragment into dodecylphosphocholine (DPC) or n-dodecyl-β-d-maltoside (DM) micelles as membrane mimics and extended our previous work on TM17 (Vincent et al., 2007, Biochim. Biophys. Acta 1768, 538). We synthesized TM16 and TM17, with the Trp residues, W1198 in TM16 and W1246 in TM17, acting as an intrinsic fluorescent probe, and TM16 and TM17 Trp variants, to probe different positions in the peptide sequence. We assessed the interaction of peptides with membrane mimics by evaluating the increase in fluorescence intensity resulting from such interactions. In all micelle-bound peptides, the tryptophan residue appeared to be located, on average, in the head group micelle region, as shown by its fluorescence spectrum. Each tryptophan residue was partially accessible to both acrylamide and the brominated acyl chains of two DM analogs, as shown by fluorescence quenching. Tryptophan fluorescence lifetimes were found to depend on the position of the tryptophan residue in the various peptides, probably reflecting differences in local structures. Far UV CD spectra showed that TM16 contained significant β-strand structures. Together with the high Trp correlation times, the presence of these structures suggests that TM16 self-association may occur at the interface. In conclusion, this experimental study suggests an interfacial location for both TM16 and TM17 in membrane mimics. In terms of overall hMRP1 structure, the experimentally demonstrated amphipathic properties of these TM are consistent with a role in the lining of an at least partly hydrophilic transport pore, as suggested by the currently accepted structural model, the final structure being modified by interaction with other TM helices.     

Structural Changes of Human Serum Albumin Induced by Cadmium Acetate

The structural changes of human serum albumin (HSA) induced by the addition of cadmium acetate were systematically investigated using UV–vis absorption, circular dichroism (CD), synchronous, and three‐dimentional (3D) fluorescence methods. The fluorescence spectra suggested the formation of cadmium acetate–HSA complex. UV absorption result indicated that the interaction between cadmium acetate and HSA could lead to the alteration of the protein skeleton. The structural analysis according to CD method showed that the cadmium acetate binding altered HSA conformation with a major reduction of α‐helix, inducing a partial protein unfolding. Synchronous fluorescence spectra suggested that cadmium acetate was situated closer to tryptophan residue compared to tyrosine residues, making tryptophan residue locate in a more hydrophobic environment. 3D fluorescence demonstrated that cadmium acetate could induce the HSA aggregation and cause a slight unfolding of the polypeptide backbone of the protein.     

pH-induced conformational states of bovine growth hormone

The folding behavior of bovine growth hormone (bGH) is examined by chemical and pH denaturation using several spectroscopic probes of protein secondary and tertiary structure. Partially denaturing concentrations of urea eliminate the native-state quenching of intrinsic tryptophan fluorescence, from the single protein tryptophan, but the fluorescence emission spectrum is not red-shifted like the unfolded state, and the protein retains substantial secondary structure. A neutral-to-acid pH shift also eliminates tryptophan quenching; however, the loss of quenching is not accompanied by an emission red-shift. In addition, the protein undergoes a pH-dependent UV absorbance transition; the changes in absorptivity have the same midpoint as the transition associated with the change in intrinsic tryptophan fluorescence. The magnitude of the absorption transition is similar to that observed previously for urea denaturation of the protein. In a similar fashion, a pH-dependent CD transition is also observed; however, the transition occurs at a higher pH. The behavior of the various optical probes indicates that the pH-induced conformational transition produces a highly populated species in which the microenvironment surrounding the single protein tryptophan residue resembles that observed during the urea-induced unfolding/refolding transition. The pH-induced changes in tertiary structure occur at a lower pH than the changes associated with a portion of the secondary structure. Proton NMR of the low-pH intermediate indicates that the three His and six Tyr resonances are indistinguishable from the unfolded state. The intermediate(s) observed by either chemical or pH-induced denaturation resemble(s) a molten globule state which contains significant secondary structure. The residual secondary structure present in the intermediate could be nonnative.(ABSTRACT TRUNCATED AT 250 WORDS)     

An unusual red-edge excitation and time-dependent Stokes shift in the single tryptophan mutant protein DD-carboxypeptidase from Streptomyces: the role of dynamics and tryptophan rotamers

The fluorescence emission of the single tryptophan (W233) of the mutant protein DD-carboxypeptidase from streptomyces is characterized by a red-edge excitation shift (REES), i.e., the phenomenon that the wavelength of maximum emission depends on the excitation wavelength. This phenomenon is an indication for a strongly reduced dynamic environment of the single tryptophan, which has a very low accessibility to the solvent. The REES shows, however, an unusual temperature and time dependence. This, together with the fluorescence lifetime analysis, showing three resolvable lifetimes, can be explained by the presence of three rotameric states that can be identified using the Dead-End Elimination method. The three individual lifetimes increase with increasing emission wavelength, indicating the presence of restricted protein dynamics within the rotameric states. This is confirmed by time-resolved anisotropy measurements that show dynamics within the rotamers but not among the rotamers. The global picture is that of a protein with a single buried tryptophan showing strongly restricted dynamics within three distinct rotameric states with different emission spectra and an anisotropic environment.     

渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦（TriticumsativumL．）根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯（DPH）荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H －AT－Pase活力降低,暗示膜蛋白的构象和功能发生了改变。结果表明,质膜膜脂物理状态改变可能是植物感受渗透胁迫的原初响应。