Intrinsic Fluorescence Spectroscopy Research Articles

Interaction Between Fluorinated Amphiphilic Copolymer P(HFMA)-g-P(SPEG) and BSA

A novel fluorinated amphiphilic copolymer P(HFMA)-g-P(SPEG) was synthesized. The interactions between P(HFMA)-g-P(SPEG) and bovine serum albumin (BSA) were studied by synchronous fluorescence and intrinsic fluorescence spectroscopy. It was concluded through synchronous fluorescence that P(HFMA)-g-P(SPEG) mainly bound to tryptophan residues of BSA. Intrinsic fluorescence results revealed that BSA and P(HFMA)-g-P(SPEG) had strong interactions. The mechanism of quenching belonged to dynamic quenching and the main sort of binding force was hydrophobic force. The hydrophobic interaction between P(HFMA)-g-P(SPEG) and BSA was conformed by micropolarity and TEM photographs.

Stability of haloalkaliphilic Geomicrobium sp. protease modulated by salt

A novel protease from the halophilic bacterium Geomicrobium sp. EMB2 (MTCC 10310) is described. The activity of the protease was modulated by salt, and it exhibited remarkable stability in organic solvents, at alkaline pH, and in other denaturing conditions. The structural changes under various denaturing conditions were analyzed by measurements of intrinsic fluorescence and circular dichroism spectroscopy. Circular dichroism showed that the secondary structure of the protease was predominantly α-helical but unfolded in salt-free medium. The structure is regained by inclusion of NaCl in the range of 2-5%. The presence of NaCl exerted a protective effect against thermal, organic solvent, and guanidine hydrochloride denaturation by preventing unfolding.

The Congenital Cataract-Linked G61C Mutation Destabilizes γD-Crystallin and Promotes Non-Native Aggregation

γD-crystallin is one of the major structural proteins in human eye lens. The solubility and stability of γD-crystallin play a crucial role in maintaining the optical properties of the lens during the life span of an individual. Previous study has shown that the inherited mutation G61C results in autosomal dominant congenital cataract. In this research, we studied the effects of the G61C mutation on γD-crystallin structure, stability and aggregation via biophysical methods. CD, intrinsic and extrinsic fluorescence spectroscopy indicated that the G61C mutation did not affect the native structure of γD-crystallin. The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding. The mutation mainly affected the transition from the native state to the intermediate but not that from the intermediate to the unfolded or aggregated states. At high temperatures, both proteins were able to form aggregates, and the aggregation of the mutant was much more serious than the wild type protein at the same temperature. At body temperature and acidic conditions, the mutant was more prone to form amyloid-like fibrils. The aggregation-prone property of the mutant was not altered by the addition of reductive reagent. These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.

Comparison of the Chaperoning Action of Glycerol and β-Casein on Aggregation of Proteins in the Presence of Crowding Agent

β-Casein is one of the major components of the milk micelles of most mammals and has been shown to exhibit in vitro chaperone-like activity. Glycerol is a chemical chaperone belonging to the polyol family, which increases protein stability and inhibits protein aggregation. These prompted us to compare the chaperone-like activity of β-casein and glycerol. In this study, the effect of β-casein and glycerol on folding of the target proteins (ovotransferrin, insulin and α-lactalbumin) in the presence of dextran, as a macromolecular crowding agent, is examined using visible absorption spectroscopy, intrinsic fluorescence spectroscopy, 1-anilino-8-naphthalene sulfonic acid fluorescence binding and near CD spectroscopy. In the presence of dextran, the rate and extent of aggregation of target proteins was enhanced and β-casein was less effective in preventing the aggregation and precipitation of target proteins. These data support the hypothesis that β-casein interacts more effectively with slowly aggregating rather than rapidly aggregating target proteins. It is proposed that dextran-induced changes to protein conformation and the rate of intermolecular association are in a kinetic competition with the chaperoning action of β-casein; however our results demonstrated the higher activity of glycerol, as a chemical chaperone, than β-casein on the folding of target proteins, especially in the presence of dextran. This is likely due to the stabilizing effect of glycerol on protein structure and environment. The implications for the in vivo functions of β-casein and glycerol, based on their exhibiting such in vitro chaperone-like activities, are discussed.

The binding of 14-3-3γ to membranes studied by intrinsic fluorescence spectroscopy

Human 14-3-3 proteins contain two conserved tryptophan residues in each monomer, Trp60 and Trp233 in isoform γ. 14-3-3γ binds to negatively charged membranes and here we show that membrane binding can be monitored by steady-state intrinsic fluorescence spectroscopy. Measurements with W60F and W233F 14-3-3γ mutants revealed that Trp60 is the major contributor to the emission fluorescence, whereas the fluorescence of Trp233, which π-stacks with Tyr184, is quenched. The fluorescence is reduced and red-shifted upon specific binding of a phosphate ligand, and further red-shifted upon binding of 14-3-3γ to the membrane, compatible with solvent exposure of Trp60. Moreover, our results support that membrane binding involves the non-conserved, convex area of 14-3-3γ, and that Trp residues do not intercalate in the bilayer.

Enhanced physical stability of human calcitonin after methionine oxidation

Calcitonin is a blood-calcium-lowering peptide, present in different species, which inhibits the resorption of bone by osteoclasts. Human calcitonin (hCT) is one of the few calcitonin peptides, which contains a methionine residue; this residue is in position 8. Methionines are known to be readily oxidized to sulfoxides both in vivo and in vitro. The current work describes the effect of methionine oxidation on the physical stability of hCT. Aggregation kinetics of human calcitonin were studied at different pH values by intrinsic fluorescence spectroscopy, turbidity at 350 nm, microscopy analyses, Nile Red, and 1,8-ANS fluorescence emission. In all the experiments, methionine oxidation reduced the aggregation rate of human calcitonin. The effect of methionine oxidation was independent of pH. Fluorescence lifetime data also showed that the conformation of hCT in the aggregated state can be influenced by methionine oxidation. A hypothesis for the enhanced physical stability of oxidized hCT is presented and discussed.

Computational Design and Biophysical Characterization of Aggregation-Resistant Point Mutations for γD Crystallin Illustrate a Balance of Conformational Stability and Intrinsic Aggregation Propensity

γD crystallin is a natively monomeric eye-lens protein that is associated with hereditary juvenile cataract formation. It is an attractive model system as a multidomain Greek-key protein that aggregates through partially folded intermediates. Point mutations M69Q and S130P were used to test (1) whether the protein-design algorithm RosettaDesign would successfully predict mutants that are resistant to aggregation when combined with informatic sequence-based predictors of peptide aggregation propensity and (2) how the mutations affected relative unfolding free energies (ΔΔG(un)) and intrinsic aggregation propensity (IAP). M69Q was predicted to have ΔΔG(un) ≫ 0, without significantly affecting IAP. S130P was predicted to have ΔΔG(un) ∼ 0 but with reduced IAP. The stability, conformation, and aggregation kinetics in acidic solution were experimentally characterized and compared for the variants and wild-type (WT) protein using circular dichroism and intrinsic fluorescence spectroscopy, calorimetric and chemical unfolding, thioflavin-T binding, chromatography, static laser light scattering, and kinetic modeling. Monomer secondary and tertiary structures of both variants were indistinguishable from WT, while ΔΔG(un) > 0 for M69Q and ΔΔG(un) < 0 for S130P. Surprisingly, despite being the least conformationally stable, S130P was the most resistant to aggregation, indicating a significant decrease of its IAP compared to WT and M69Q.

Detection of coronary atherosclerotic plaques with superficial proteoglycans and foam cells using real-time intrinsic fluorescence spectroscopy

ObjectivesThe protein components of low-density lipoprotein (LDL), oxidized LDL and proteoglycans such as versican contain tryptophan, an amino acid with characteristic fluorescence features at 308nm excitation wavelength. We hypothesize that intrinsic fluorescence spectroscopy at 308nm excitation wavelength IFS308, a method suitable for clinical use, can identify coronary artery lesions with superficial foam cells (SFCs) and/or proteoglycans. MethodsWe subjected 119 human coronary artery specimens to in vitro fluorescence and reflectance spectroscopy. We used 5 basis spectra to model IFS308, and extracted their contributions to each individual IFS308 spectrum. A diagnostic algorithm using the contributions of Total Tryptophan and fibrous cap to IFS308 was built to identify specimens with SFCs and/or proteoglycans in their top 50μm. ResultsWe detected SFCs and/or proteoglycans, such as versican or the glycosaminoglycan hyaluronan, in 24 fibrous cap atheromas or pathologic intimal thickening (PIT) lesions. An algorithm using the contributions of Total Tryptophan and fibrous cap to IFS308 was able to identify these segments with 92% sensitivity and 80% specificity. ConclusionWe were able to establish a set of characteristic LDL, oxidized LDL, versican and hyaluronan fluorescence spectra, ready to be used for real-time diagnosis. The IFS308 technique detects SFCs and/or proteoglycans in fibrous cap atheromas and PIT lesions. SFCs and proteoglycans are histological markers of vulnerable plaques, and this study is a step further in developing an invasive clinical tool to detect the vulnerable atherosclerotic plaque.

Purification of Recombinant Acyl-Coenzyme A:Cholesterol Acyltransferase 1 (ACAT1) from H293 Cells and Binding Studies between the Enzyme and Substrates Using Difference Intrinsic Fluorescence Spectroscopy

Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a membrane-bound enzyme utilizing long-chain fatty acyl-coenzyme A and cholesterol to form cholesteryl esters and coenzyme A. Previously, we had expressed tagged human ACAT1 (hACAT1) in CHO cells and purified it to homogeneity; however, only a sparse amount of purified protein could be obtained. Here we report that the hACAT1 expression level in H293 cells is 18-fold higher than that in CHO cells. We have developed a milder purification procedure to purify the enzyme to homogeneity. The abundance of the purified protein enabled us to conduct difference intrinsic fluorescence spectroscopy to study the binding between the enzyme and its substrates in CHAPS/phospholipid mixed micelles. The results show that oleoyl-CoA binds to ACAT1 with K(d) = 1.9 μM and elicits significant structural changes of the protein as manifested by the significantly positive changes in its fluorescence spectrum; stearoyl-CoA elicits a similar spectrum change but much lower in magnitude. Previously, kinetic studies had shown that cholesterol is an efficient substrate and an allosteric activator of ACAT1, while its diastereomer epicholesterol is neither a substrate nor an activator. Here we show that both cholesterol and epicholesterol induce positive changes in the ACAT1 fluorescence spectrum; however, the magnitude of spectrum changes induced by cholesterol is much larger than epicholesterol. These results show that stereospecificity, governed by the 3β-OH moiety in steroid ring A, plays an important role in the binding of cholesterol to ACAT1.

The role of tyrosine 207 in the reaction catalyzed by Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.

The functional significance of tyrosine 207 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase was explored by examining the kinetic properties of the Tyr207Leu mutant. The variant enzyme retained the structural characteristics of the wild-type protein as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analyses of the mutated variant showed a 15-fold increase in K(m)CO₂, a 32-fold decrease in V(max), and a 6-fold decrease in K(m) for phosphoenolpyruvate. These results suggest that the hydroxyl group of Tyr 207 may polarize CO₂ and oxaloacetate, thus facilitating the carboxylation/decarboxylation steps.

Thermally responsive graft copolymer of soy protein isolate and N-isopropylacrylamide: synthesis and self-assembly behavior in aqueous solution

In recent years, soy protein isolate (SPI) has attracted great attention due to its biodegradability, biocompatibility, and wide availability. It has been used in food and pharmaceutical industry such as edible films and drug delivery systems. In this study, we report the synthesis and self-assembly behavior in aqueous solution of thermally responsive graft copolymer (SPI-g-poly(N-isopropylacrylamide) (PNIPA)) of soy protein isolate and N-isopropylacrylamide in aqueous solution. SPI-g-PNIPA was synthesized in the 8 mol/l urea cushioning solution, by using ammonium persulfate as the initiator and mercaptoacetic acid as the protein unfolding agent. Laser light scattering, transmission electron microscopy, and fluorescence spectroscopy have been used to study the self-assembly behavior of SPI-g-PNIPA in aqueous solution. Above the critical micelle concentration (cmc), SPI-g-PNIPA aggregates could assemble into different structures including the simple spherical structure, spherical core–shell structure, and random coil structure, depending on the graft copolymer concentration. The graft copolymer concentration, temperature, pH value, and ionic strength were found to influence the aggregate size and morphology of SPI-g-PNIPA in aqueous solution. With increasing ionic strength, the aggregate size increases. However, pH value, SPI-g-PNIPA concentration, and temperature have complicated influences on the aggregate size. The lower critical solution temperature of the SPI-g-PNIPA at pH 8.5 is 36 °C. The method of intrinsic fluorescence spectroscopy was used for the first time to determine the cmc value of SPI-g-PNIPA in aqueous solution.

Sequential Events in the Irreversible Thermal Denaturation of Human Brain-Type Creatine Kinase by Spectroscopic Methods

The non-cooperative or sequential events which occur during protein thermal denaturation are closely correlated with protein folding, stability, and physiological functions. In this research, the sequential events of human brain-type creatine kinase (hBBCK) thermal denaturation were studied by differential scanning calorimetry (DSC), CD, and intrinsic fluorescence spectroscopy. DSC experiments revealed that the thermal denaturation of hBBCK was calorimetrically irreversible. The existence of several endothermic peaks suggested that the denaturation involved stepwise conformational changes, which were further verified by the discrepancy in the transition curves obtained from various spectroscopic probes. During heating, the disruption of the active site structure occurred prior to the secondary and tertiary structural changes. The thermal unfolding and aggregation of hBBCK was found to occur through sequential events. This is quite different from that of muscle-type CK (MMCK). The results herein suggest that BBCK and MMCK undergo quite dissimilar thermal unfolding pathways, although they are highly conserved in the primary and tertiary structures. A minor difference in structure might endow the isoenzymes dissimilar local stabilities in structure, which further contribute to isoenzyme-specific thermal stabilities.

A comparative study of physicochemical and conformational properties in three vicilins from Phaseolus legumes: Implications for the structure–function relationship

The polypeptide constituents, physicochemical and conformational properties of three vicilins from kidney, red and mung beans, fractionated using combined acid extraction and ion-exchange chromatographic techniques, were characterized and compared. The tested physicochemical and conformational properties included amino acid composition, surface charge and hydrophobicity, protein solubility (PS; as a function of pH), emulsifying activity index (EAI) and emulsion stability index (ESI), as well as secondary, tertiary and/or quaternary conformations. The amino acid composition (especially relative acidic/basic charged amino acid ratio, and the percentage of polar uncharged amino acids) was an intrinsic physicochemical parameter that largely determined other physicochemical and conformational properties. The PS and EAI (and ESI) of these vicilins at various pH values were associated with each other, and both were closely related to their surface charge and/or hydrophobicity. Far-UV CD spectral analyses indicated that these vicilins exhibited similar secondary structure compositions at pH deviating from the isoelectric point (about pH 4.5). However, their tertiary and/or quaternary conformations, as determined by differential scanning calorimetry, intrinsic fluorescence and near-UV CD spectroscopy, remarkably varied. At pH 7.0 or 9.0, there were close relationships between the EAI and the flexibility in quaternary conformation, and between the ESI and the flexibility in tertiary conformation. This is the first observation to elucidate the importance of tertiary and/or quaternary conformations for the emulsifying properties of legume vicilins. Additionally, an underlying mechanism of the emulsification process for the vicilins (trimeric form) with excellent emulsifying properties was proposed.

The Effects of Substituted Cyclodextrins on the Colloidal and Conformational Stability of Selected Proteins

The effects of various types of substituted and nonsubstituted cyclodextrins (CDs) on the physical and colloidal stability of three different proteins were studied to further ascertain the mechanism by which cyclodextrins stabilize proteins. The three proteins examined in this study are the Clostridium difficile Toxoid A, Yersinia pestis low-calcium-response V or V antigen (LcrV), and fibroblast growth factor 10 (FGF-10). These three pharmaceutically relevant proteins differ in molecular weight, pI, as well as in their secondary and tertiary structure. The effects of three parent cyclodextrins (alpha, beta, and gamma), as well as several hydroxypropyl (HP-CDs) and sulfobutylether (SBE-CDs) cyclodextrins of varying degrees of substitution, on the three proteins were examined as a function of pH and temperature. Structural changes and aggregation behavior were monitored in the presence and absence of the 17 cyclodextrins using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering. Overall, the major effect of the cyclodextrins on the proteins was the ability of a majority of them to inhibit thermally induced aggregation. This study suggests that the stabilization of proteins by cyclodextrins is dictated by their type and degree of substitution, as well as the physical and chemical properties of the protein being examined.

Stability of Pin1 as revealed by thermal and spectroscopic studies

Pin1 is a two-domain enzyme which has peptidyl–prolyl cis/trans isomerase activity. Pin1 recognizes phospho-Ser/Thr-Pro motifs in cell-signaling proteins, and is both a cancer and an Alzheimer’s disease target. The thermal stability of Pin1 was studied intensively by SDS–PAGE, enzymatic activity assay, intrinsic fluorescence spectroscopy and circular dichroism spectroscopy. The activity of Pin1 gradually decreased above 40 °C, and the Tm was 57.6 ± 1.0 °C. Fluorescence experiments indicated that heat treatment induced changes in the substructures in Pin1, resulting in that the polarity in the microenvironments of the tryptophan residues increased. It is assumed that the thermal denaturation of Pin1 involved a three-state transition. The intermediate state of Pin1 at about 60 °C was confirmed by fluorescence emission spectra, the synchronous fluorescence spectra and CD measurements. Decreases in α-helix and β-sheet appeared above 40 °C, which was balanced by an enhancement in unordered coil. The Tm values calculated from α-helix transition and β-sheet transition were 54.6 ± 0.6 °C and 70.7 ± 3.3 °C, respectively. Our results illustrated that Pin1 had a relatively high thermal stability and the WW domain had a higher stability than the PPIase domain.

When Reduction Leads to Expansion: A Simple Native PAGE Gel Enzyme Assay Experiment as a Starting Point for Other Structure‐Function Investigations

A simple assay on native PAGE gel for two E. coli enzymes, purified by undergraduates in a biochemistry laboratory, provides an excellent springboard for more sophisticated investigations on protein structure. They perform colorimetric assays on b‐galactosidase (b‐gal) using ortho‐nitrophenyl‐b‐galactosidase in a buffer containing b‐mercaptoethanol (BME) and on alkaline phosphatase (AP) using FAST RED TR/Naphthol AS‐MX reagent. Half of the class assays for b‐gal first and AP second; the other half of the class reverses the order. Results differ because the former group reduces two disulfide bonds which are essential for AP activity, only observing activity for b‐gal, while the latter sees activity for both enzymes.This simple PAGE experiment leads the students to the question: Is loss of activity due to protein denaturation? Answering this question raises more sophisticated investigations, including BME dialysis, intrinsic fluorescence spectroscopy of AP tryptophan residues, ANS fluorescence spectroscopy, and circular dichroism. These can be offered as special projects to laboratory groups who report their findings to the entire class, providing a holistic approach to investigating the relationship between loss of activity and protein denaturation. Initial data and results from our laboratory course will be presented. This work supported in part by a grant from NSF (DUE 0837398 to JTH).

Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2)

The influence of two different pH values on the physical stability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in aqueous solution was evaluated in the present work. RhBMP-2 in solution at pH 4.5 or 6.5 was characterized by intrinsic and extrinsic (Nile Red and 1,8-ANS) fluorescence spectroscopy, 90° light-scattering and transmission electron microscopy (TEM). Compared to the pH 4.5 solution, rhBMP-2 at pH 6.5 had (i) a stronger intrinsic fluorescence intensity, (ii) a longer fluorescence lifetime, (iii) a stronger 90° light-scattering intensity, (iv) a stronger Nile Red fluorescence intensity, (v) a higher Nile Red fluorescence anisotropy, (vi) a lower 1,8-ANS fluorescence intensity, (vii) a higher 1,8-ANS fluorescence anisotropy and (viii) a longer 1,8-ANS fluorescence lifetime. Electron microscopy showed that rhBMP-2 at pH 4.5 contained aggregates of about 100 nm in diameter. More and larger protein aggregates (0.1–2 μm) were observed in solution at pH 6.5. Taken together, these results indicate conformational changes and increased aggregation of rhBMP-2 at pH 6.5 compared to pH 4.5, demonstrating a strong influence of pH on rhBMP-2 physical stability. These observations must be considered when developing a delivery system for rhBMP-2.

Equilibrium and kinetics of the unfolding and refolding of Escherichia coli Malate Synthase G monitored by circular dichroism and fluorescence spectroscopy

The equilibrium and kinetics studies of an 82 kDa large monomeric Escherichia coli protein Malate Synthase G (MSG) was investigated by far and near-UV CD, intrinsic tryptophan fluorescence and extrinsic fluorescence spectroscopy. We find that despite of its large size, folding is reversible, in vitro. Equilibrium unfolding process of MSG exhibited three-state transition thus, indicating the presence of at least a stable equilibrium intermediate. Thermodynamic parameters suggest this intermediate resembles the unfolded state. However, the equilibrium intermediate exhibits pronounced secondary structure as measured by far-UV CD, partial tertiary structure as delineated by near-UV CD, compactness (m value) and exposed hydrophobic surface area as assessed by ANS binding, typically depicting a molten globule state. The stopped-flow kinetic data provide clear evidence for the presence of a burst phase during the refolding pathway due to the formation of an early Intermediate, within the dead time of the instrument. Refolding from 4 M to various lower concentrations until 0.4 M of GdnHCl follow biphasic kinetics at lower concentrations of GdnHCl (<0.8 M), whereas monophasic kinetics at concentrations above 1.5 M. Also, rollover in the refolding and unfolding limbs of chevron plot verifies the presence of a fast kinetic intermediate at lower concentration of GdnHCl. Based upon the above observations we hereby propose the folding pathway of a large multi-domain protein Malate Synthase G.

The Role of Tyrosine 207 in the Reaction Catalyzed by Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

The functional significance of tyrosine 207 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase was explored by examining the kinetic properties of the Tyr207Leu mutant. The variant enzyme retained the structural characteristics of the wild-type protein as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analyses of the mutated variant showed a 15-fold increase in K(m)CO₂, a 32-fold decrease in V(max), and a 6-fold decrease in K(m) for phosphoenolpyruvate. These results suggest that the hydroxyl group of Tyr 207 may polarize CO₂ and oxaloacetate, thus facilitating the carboxylation/decarboxylation steps.

10.1007/s11171-008-2004-0

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions for isolation and refolding of recombinant monomer and porin trimer were selected. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that recombinant porins are similar in the composition of secondary structure elements to isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins.