Intrinsic Fluorescence Spectroscopy Research Articles

Subsequent events to GTP binding by the plant PsbO protein: Structural changes, GTP hydrolysis and dissociation from the photosystem II complex

Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409–1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the β-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn 2+ and Ca 2+ ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.

Thermodynamic analysis of three state denaturation of Peanut Agglutinin

Peanut Agglutinin (PNA) is a homotetrameric protein with a very unusual open quaternary structure. During denaturation, it first dissociates into a molten globule like state, which subsequently undergoes complete denaturation. Urea denaturation of PNA at neutral pH has been studied by intrinsic fluorescence spectroscopy and has been fitted to a three state model, A4 <=> 4I <=> 4U, to get all the relevant thermodynamic parameters. Urea denaturation leads to continuous red shift of wavelength maxima. The molten globule like state is formed in a short range of urea concentration. Refolding of the denatured PNA has been attempted by intrinsic fluorescence study. Refolding by instantaneous dilution shows the occurrence of the formation of an intermediate at a relatively rapid rate, within few seconds. The transition from PNA tetramer to molten globule like state is found to have a DeltaG value of approximately 33 kcal/mole while it is approximately 8 kcal/mole for the transition from molten globule like state to a completely denatured state. This in turn indicates that the tetramerization in PNA contributes significantly to the stability of the oligomer.

Reduction of the Ganglioside Binding Activity of the Tetanus Toxin H C Fragment Destroys Immunogenicity: Implications for Development of Novel Tetanus Vaccines

In this study, the immunogenicities of the nontoxic H(C) fragment of tetanus toxin and derivatives lacking ganglioside binding activity were compared with that of tetanus toxoid after subcutaneous immunization of mice. Wild-type H(C) (H(C)WT) protein and tetanus toxoid both elicited strong antibody responses against toxoid and H(C) antigens and provided complete protection against toxin challenge. Mutants of H(C) containing deletions essential for ganglioside binding elicited lower responses than H(C)WT. H(C)M115, containing two amino acid substitutions within the ganglioside binding site, provided reduced protection against tetanus toxin challenge compared with H(C)WT, consistent with lower anti-H(C) and anti-toxoid antibody titers. Circular-dichroism spectroscopy and intrinsic fluorescence spectroscopy showed minimal structural perturbation in H(C)M115. We conclude that the presence of the ganglioside binding site within H(C) may be essential for induction of a fully protective anti-tetanus response comparable to that induced by tetanus toxoid by subcutaneous injection.

Spectroscopic and Calorimetric Analyses of Invasion Plasmid Antigen D (IpaD) from Shigella flexneri Reveal the Presence of Two Structural Domains

Shigella flexneri is a facultative intracellular pathogen that causes severe gastroenteritis in humans. Invasion plasmid antigen D (IpaD) is an essential participant in Shigella invasion of intestinal cells, but no detailed structural information is available to help understand the proposed role of IpaD in invasion or its interaction with other invasion proteins. Therefore, the secondary and tertiary structure and thermal stability of IpaD as well as selected IpaD deletion mutants were investigated using Fourier transform infrared (FTIR), circular dichroism (CD), and both intrinsic and extrinsic fluorescence spectroscopies. The energetics of thermal unfolding were also evaluated by differential scanning calorimetry (DSC). Secondary-structure analysis by CD and FTIR suggests that that IpaD is primarily alpha-helical with characteristics of a intramolecular coiled coil. Thermal studies revealed that the unfolding of IpaD is a complex process consisting of two transitions centered near 59 and 80 degrees C. A comparison of the data obtained with the intact protein and selected deletion mutants indicated that the lower temperature transition is a reversible event attributable to the unfolding of a small domain located at the N terminus of IpaD. In contrast, the thermal unfolding of the proposed major and highly stable C-terminal domain was irreversible and led to protein aggregation. When the results are taken together, they strongly support the idea that IpaD has two independent folding domains.

Spectroscopic studies of stability of papain in aqueous organic solvents

The effects of different concentrations of the water-miscible organic solvents ethanol, 1,4-dioxane, tetrahydrofuran (THF) and acetonitrile (ACN) on the stability of papain in aqueous solution were studied. Papain exhibited high stability in aqueous ACN, ethanol and 1,4-dioxane. Decreases in the activity of the enzyme were observed at organic solvent concentrations above 60%. THF caused a dramatic reduction in activity even at low concentrations (5–10%). The solvent-induced structural changes were followed by means of circular dichroism (CD) and intrinsic fluorescence spectroscopy measurements. The decreases in enzyme activity at 90% THF or 90% 1,4-dioxane were accompanied by the loss of the tertiary structure. However, at 90% ethanol and 90% ACN, papain exhibited an increased amount of the α-helical conformation, with little change in the tertiary structure.

Intrinsic Fluorescence and Diffuse Reflectance Spectroscopy Identify Superficial Foam Cells in Coronary Plaques Prone to Erosion

Foam cells perform critical functions in atherosclerosis. We hypothesize that coronary segments with superficial foam cells (SFCs) situated in a region of interest with a depth of 200 mum can be identified using intrinsic fluorescence spectroscopy (IFS) and diffuse reflectance spectroscopy (DRS). This is a key step in our ongoing program to develop a spectroscopic technique for real-time in vivo diagnosis of vulnerable atherosclerotic plaque. We subjected 132 human coronary segments to in vitro IFS and DRS. We detected SFCs in 13 thick fibrous cap atheromas and 8 pathologic intimal thickening (PIT) lesions. SFCs colocalized with accumulations of smooth muscle cells and proteoglycans, including hyaluronan (P<0.001). Two spectroscopic parameters were generated from analysis of IFS at 480 nm excitation and DRS. A discriminatory algorithm using these parameters identified specimens with SFC area >40%, 20%, 10%, 5%, 2.5%, and 0% of the region of interest with 98%, 98%, 93%, 94%, 93%, and 90% accuracy, respectively. Our combined IFS and DRS technique accurately detects SFCs in thick fibrous cap atheromas and PIT lesions. Because SFCs are associated with histological markers of plaque erosion, our spectroscopic technique could prove useful in identifying vulnerable plaques.

Stepwise modification of lysine residues of glucose oxidase with citraconic anhydride

Structural properties of modified forms of glucose oxidase made by stepwise specific modification have been investigated. By a single step modification, one of the modified forms resulted in the conversion of native structure of glucose oxidase to molten globule like form [S. Hosseinkhani, B. Ranjbar, H. Naderi-Manesh, M. Nemat-Gorgani, FEBS Lett. 561 (2004) 213–216]. Chemical modification of lysine residues in glucose oxidase was carried out using different concentration of citraconic anhydride. Modification brought about changes in the tertiary structure with some degree of alteration in secondary structure. FTIR, far and near-UV CD spectropolarimetry, intrinsic and extrinsic fluorescence spectroscopy showed structural changes of glucose oxidase in a concentration dependent manner. This was supported by comparative study of secondary and tertiary structure.

Coordinated Regulation of Hepatic Glucose Disposal

The objective of these studies was to elucidate the molecular mechanisms that underlie the acute response of the fasting liver to refeeding. Our focus was on two major regulators of liver metabolism, glucokinase (GK) and 6-phosphofructo 2-kinase/Fructose 2,6-bisphosphatase (PFK-2/FBP-2). We detailed the binding of these enzymes with pull-down assays, fluorescence spectroscopy, and steady state kinetics. The results have confirmed that the enzymes bind. The formation of this protein:protein complex activates GK and produces a PFK-2/FBP-2 that has a higher kinase-to-bisphosphatase activity ratio (K/B) which will act to increase the level of fructose-2,6-bisphosphate (F-2,6-P2). Therefore, we conclude that the binding of GK and PFK-2/FBP-2 provides for a coordinated up-regulation of hepatic glucose disposal because it activates both the phosphorylation of glucose (GK) and the activation of the committing step to glycolysis (6-phosphofructo 1-kinase) via the biofactor F-2,6-P2. Our results have begun to reveal the molecular mechanisms by which the enzyme activities are modulated. Intrinsic protein fluorescence spectroscopy showed that binding of GK to PFK-2/FBP-2, or the FBP-2 domain, promotes the binding of glucose to GK, thus activating it. The mechanism for the increased K/B of PFK-2/FBP-2 is less clear, yet it appears to involve at least one other liver protein. On this basis we have developed a comprehensive model of how these enzymes respond to the extra-hepatic signals generated by meal consumption that is completely consistent with previously published reports concerning GK and PFK-2/FBP-2. Funding: VA Merit Award to DAO.

Front-Face Fluorescence Spectroscopy as a Rapid and Nondestructive Tool for Differentiating Various Cereal Products: A Preliminary Investigation

The potential of intrinsic fluorescence spectroscopy was investigated for differentiating between processed grains (flours, pasta, and semolinas) of different wheat cereal products. A total of 59 samples (e.g., 20 complete Kamut, semicomplete Kamut, and soft wheat flours, 28 pasta, and 11 semolinas manufactured from complete Kamut, semicomplete Kamut, and hard wheat flours) were analyzed by front-face fluorescence spectroscopy. Tryptophan fluorescence spectra were scanned between 305 and 400 nm on samples following excitation at 290 nm. The principal component analysis (PCA) performed on flour spectra clearly differentiated complete Kamut and semicomplete Kamut samples from those produced from complete and semicomplete soft wheat flours. The PCA performed on pasta spectra discriminated samples manufactured from complete Kamut and complete hard wheat flours from those made with semicomplete Kamut and semicomplete hard wheat flours. The best discrimination was obtained from tryptophan spectra recorded on semolinas since the four groups were well discriminated. Correct classification amounting to 61.9% was obtained for pasta spectra. A better classification was obtained for flour and semolina spectra since correct classification amounted to 86.7% and 87.9%, respectively. Front-face fluorescence spectroscopy has the potential to be a rapid, low-cost, and efficient method for the authentication of cereal products.

Examination of the Slow Unfolding of Pro-Nerve Growth Factor Argues against a Loop Threading Mechanism for Nerve Growth Factor

Nerve growth factor (NGF), a member of the neurotrophin family, is an all-beta-sheet protein with a characteristic structure motif, the cystine knot. Unfolding of NGF in 6 M GdnHCl has been described previously to involve an initial partial loss of structure and a subsequent very slow conversion to a second, completely unfolded state. This latter conversion was postulated to represent a back-threading of the disulfide bond that passes through the cystine knot (loop threading hypothesis). Here, this hypothesis was questioned with the pro form of the protein (proNGF). In proNGF, the mature part is preceded by the 103-amino acid pro-peptide. Consequently, loop threading of the N-terminally extended protein should be significantly delayed. However, unfolding kinetics of proNGF monitored by RP-HPLC, intrinsic fluorescence, and NMR spectroscopy were comparable to those of mature NGF. Time-resolved (1)H-(15)N HSQC spectra revealed a slow time-dependent loss of residual structure of which the kinetics correlated well with the transition observed by RP-HPLC. Refolding from the completely unfolded state led to a partial recovery of natively folded proNGF. In summary, the sequential unfolding of proNGF only marginally differed from that of mature NGF. Therefore, it is very unlikely that a loop threading mechanism is the cause of the slow unfolding step.

Detection of morphological markers of vulnerable atherosclerotic plaque using multimodal spectroscopy

Vulnerable plaques, which are responsible for most acute ischemic events, are presently invisible to x-ray angiography. Their primary morphological features include a thin or ulcerated fibrous cap, a large necrotic core, superficial foam cells, and intraplaque hemorrhage. We present evidence that multimodal spectroscopy (MMS), a novel method that combines diffuse reflectance spectroscopy (DRS), intrinsic fluorescence spectroscopy (IFS), and Raman spectroscopy (RS), can detect these markers of plaque vulnerability. To test this concept, we perform an MMS feasibility study on 17 human carotid artery specimens. Following the acquisition of spectra, each specimen is histologically evaluated. Two parameters from DRS, hemoglobin concentration and a scattering parameter, are used to detect intraplaque hemorrhage and foam cells; an IFS parameter that relates to the amount of collagen in the topmost layers of the tissue is used to detect the presence of a thin fibrous cap; and an RS parameter related to the amount of cholesterol and necrotic material is used to detect necrotic core. Taken together, these spectral parameters can generally identify the vulnerable plaques. The results indicate that MMS provides depth-sensitive and complementary morphological information about plaque composition. A prospective in vivo study will be conducted to validate these findings.

Structural modulation of calmodulin and calmodulin-dependent protein kinase II by pea protein hydrolysates

The effects of two fractions of pea protein hydrolysate with high levels of positively charged amino acids on the structural conformations of calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) were determined using fluorescence and circular dichroism methods. In the presence of Ca2 + , addition of the protein hydrolysates to CaM and CaM/CaMKII complex led to increased exposure of aromatic groups as measured by intrinsic and extrinsic fluorescence spectroscopy. Near-UV circular dichroism data revealed an increase in the tertiary structure of CaM in the presence of pea protein hydrolysates. Effect of the protein hydrolysates on the CaM structure was greater with the fraction that contained higher contents of arginine and lysine when compared with the fraction with lower levels of these two amino acids. We concluded that the presence of the pea protein hydrolysates led to rearrangement of the native protein structure and exposure of buried hydrophobic groups of CaM and/or CaMKII.

Characterization of the Interaction of Single Tryptophan Containing Mutants of IpaC from Shigella flexneri with Phospholipid Membranes

Shigella flexneri causes dysentery after invading the epithelial cells of the human colon. Enterocyte invasion is induced by the bacterial effector IpaC (invasion plasmid antigen C), which triggers Shigella entry into epithelial cells by a rather poorly understood mechanism. IpaC is also involved in pathogen escape into the host cell cytoplasm following uptake, and this property may be reflected in its ability to disrupt phospholipid vesicles in vitro. Purified recombinant IpaC interacts with liposome vesicles to cause the release of small molecules trapped inside. This interaction requires that the liposomes possess an acidic phospholipid component. To better understand the events involved in the disruption of liposomes by IpaC, single tryptophan mutants were generated to permit the use of intrinsic fluorescence, circular dichroism, and ultraviolet absorption spectroscopies to examine the effect that phospholipid membrane association has on IpaC structure and stability. These mutants were also used to determine how amino acid substitutions within specific regions of IpaC influence its activity in vivo. The outcomes of this study include findings that cholesterol greatly impacts IpaC association with phospholipid membranes, tryptophan incorporation into specific regions of IpaC (especially near the C-terminus) can greatly impact its in vivo activity, and interaction with phospholipid membranes causes differing degrees of change in the fluorescence of tryptophan residues introduced at specific sites within IpaC. These data, together with fluorescence quenching analyses, provide new functional and structural information concerning IpaC and its insertion into phospholipid membranes.

Characterization of the Redox and Metal Binding Activity of BsSco, a Protein Implicated in the Assembly of Cytochrome c Oxidase

Members of the Sco protein family are implicated in the assembly of the respiratory complex cytochrome c oxidase. Several possible roles have been proposed for Sco: a copper delivery agent, a site-specific thiol reductase, and an indicator of cellular redox status. Two cysteine residues (C45 and C49) in the sequence CXXXCP and a histidine (H135) approximately 90 residues toward the C-terminus are conserved in Sco from bacteria, yeast, and humans. The soluble domain of Sco has a thioredoxin fold that is suggestive of redox activity for this protein. We have characterized the soluble domain of the Sco protein from Bacillus subtilis (i.e., sBsSco) for its redox reactivity and metal binding capacity. In oxidized sBsSco, the cysteines are present as an intramolecular disulfide. Oxidized sBsSco does not bind metal, but can be reduced in vitro to a metal-binding form. Reduction of the disulfide in sBsSco is accompanied by increased intrinsic fluorescence. The reducibility of the cystine is unchanged when the conserved histidine is mutated to alanine. Tight binding by reduced sBsSco is observed for Cu(II) by electronic absorption, intrinsic fluorescence, and EPR spectroscopies, and isothermal titration calorimetry with an observed stoichiometry of one Cu(II) ion per sBsSco and a KD of approximately 50 nM. Tight binding of Cu(I) and Ag(I) is observed by quenching of intrinsic tryptophan fluorescence. Cobalt(II) exhibits weak binding, whereas Ni(II) and Zn(II) do not appear to bind. The high-affinity binding of metals by BsSco is triggered by its redox state, and this property could be important for its function in vivo.

The effect of heparin on structural and functional properties of low density lipoproteins

Heparin binding to human low density lipoproteins (LDL) and the effect of heparin on the ability of LDL to bind to the LDL receptor has been investigated. Emphasis has been made on the physiological conditions of temperature, pH and the ionic strength. Intrinsic fluorescence spectroscopy of LDL has been applied to follow heparin binding. Fluorescence anisotropy has been measured to describe the changes in apoB and dansyl–heparin dynamics upon binding. Eu 3+-labeled LDL binding to the intact LDL receptor has been monitored by time-resolved fluorescence spectroscopy technique. We have found that heparin binds to LDL under the physiological conditions, probably by Van der Waals interactions and hydrogen bonding. Temperature seems to be the most important factor influencing the interaction. Furthermore, the presence of heparin inhibits LDL binding to the intact LDL receptor that might have consequences on the cholesterol metabolism in vivo.

Activity and structural changes of mushroom tyrosinase induced by n-alkyl sulfates

Catecholase activity and structural changes of mushroom tyrosinase (MT) were studied in the presence of some n-alkyl sulfate derivatives. Experiments showed that MT reached its optimal activity in the presence of 1.5, 0.6, and 0.2 mM of sodium n-octyl sulfate (SOS), sodium n-dodecyl sulfate (SDS) and sodium n-tetradecyl sulfate (STS), respectively. Native and incubated MT with the n-alkyl sulfates were also investigated from structural point of view by far-UV circular dichroism (CD) and intrinsic fluorescence spectroscopy. At the above mentioned concentrations of SOS, SDS, and STS no change in the secondary structure of MT was observed. However, changes in the tertiary structure of the enzyme due to the presence of n-alkyl sulfates were obvious. Results of this research indicate that n-alkyl sulfate with longer chain induces greater conformational changes in MT, hence, can activate it at lower concentrations.

The C‐terminal Ca2+‐binding domain of SPARC confers anti‐spreading activity to human urothelial cells

The anti-spreading activity of secreted protein acidic and rich in cysteine (SPARC) has been assigned to the C-terminal third domain, a region rich in alpha-helices. This "extracellular calcium-binding" (EC) domain contains two EF-hands that each coordinates one Ca2+ ion, forming a helix-loop-helix structure that not only drives the conformation of the protein but is also necessary for biological activity. Recombinant (r) EC, expressed in E. coli, was fused at the C-terminus to a His hexamer and isolated under denaturing conditions by nickel-chelate affinity chromatography. rEC-His was renatured by procedures that simultaneously (i) removed denaturing conditions, (ii) catalyzed disulfide bond isomerization, and (iii) initiated Ca2+-dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC-His exhibited a Ca2+-dependent conformation that was consistent with the known crystal structure. Spreading assays confirmed that rEC-His was biologically active through its ability to inhibit the spreading of freshly plated human urothelial cells propagated from transitional epithelium. rEC-His and rSPARC-His exhibited highly similar anti-spreading activities when measured as a function of concentration or time. In contrast to the wild-type and EC recombinant proteins, rSPARC(E268F)-His, a point substitution mutant at the Z position of EF-hand 2, failed to exhibit both Ca2+-dependent changes in alpha-helical secondary structure and anti-spreading activity. The collective data provide evidence that the motif of SPARC responsible for anti-spreading activity was dependent on the coordination of Ca2+ by a Glu residue at the Z position of EF-hand 2 and provide insights into how adhesive forces are balanced within the extracellular matrix of urothelial cells. .

Periplasmic Protein HdeA Exhibits Chaperone-like Activity Exclusively within Stomach pH Range by Transforming into Disordered Conformation

The extremely acidic environment of the mammalian stomach, with a pH range usually between 1 and 3, represents a stressful challenge for enteric pathogenic bacteria such as Escherichia coli before they enter into the intestine. The hdeA gene of E. coli was found to be acid inducible and was revealed by genetic studies to be important for the acid survival of the strain. This study was performed in an attempt to characterize the mechanism of the activity of the HdeA protein. Our data provided in this report strongly suggest that HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3). Furthermore, our data indicate that HdeA exposes hydrophobic surfaces that appear to be involved in the binding of denatured substrate proteins at extremely low pH values. In light of our observations, models are proposed to explain the action of HdeA in both a physiological and a molecular context.

Linking tricyclic antidepressants to ionotropic glutamate receptors

Although tricyclic antidepressants have been in existence since the 1940s when they were discovered upon screening iminodibenzyl derivatives for other potential therapeutic uses, their mechanism of action has remained unclear [A. Goodman Gilman, T.W. Rall, A.S. Nies, P. Taylor, Goodman and Gilman’s The Pharmacological Basis of Therapeutics, eighth ed., Pergamon Press, New York, 1990]. In addition to their ability to hinder the reuptake of biogenic amines, there is mounting evidence that the tricyclic antidepressants inhibit glutamate transmission. Here, intrinsic tryptophan fluorescence spectroscopy is used to document the binding of desipramine, a member of the tricyclic antidepressant family, to a well-defined extracellular glutamate binding domain (S1S2) of the GluR2 subunit of the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. The binding is distinct from those of other known effectors of the receptor, including the endogenous sulfated neurosteroids pregnenolone sulfate and 3α-hydroxy-5β-pregnan-20-one sulfate, and is consistent with a conformational change upon binding that is allosterically transmitted to the channel region of the receptor.

Effect of caffeine on oxidation susceptibility of human plasma low density lipoproteins

The effect of caffeine on oxidation susceptibility of low density lipoproteins (LDL) has been studied. LDL oxidation was induced by copper ions and an azo initiator. The conjugated dienes formation was followed spectrophotometrically and indicated the LDL oxidation status. Changes in LDL protein moiety during the lag phase, studied only in the experiments of copper induced oxidation, were followed using the intrinsic fluorescence spectroscopy. The decay of LDL fluorescence signal during initial stages of oxidation was slower in the presence of caffeine. Supported by the fluorescence quenching and polarization measurements, these results may indicate the protective role of caffeine against LDL oxidation in vitro. The results also indicate that the production of conjugated dienes in the propagation and decomposition phase of LDL oxidation is lower in the presence of caffeine, regardless of the initiation mechanism. These findings may have implications for the effect of caffeine on LDL in vivo.