Spectroscopic investigation of the effects of aqueous‐phase prepared CdTe quantum dots on protein hemoglobin at the molecular level

3‐Mercaptopropionic Acid‐modified CdTe quantum dots (QDs) were synthesized and characterized by infrared, fluorescence, and ultraviolet–visible absorption spectra and Nano‐ZetaSizer measurements. Then the interaction between QDs and hemoglobin was studied to investigate the effects of QDs on the structure and function of hemoglobin by using a variety of spectroscopy methods and isothermal titration calorimetry. The results showed van der Waals forces and hydrogen bonding predominantly played major roles in the binding. The intrinsic fluorescence of hemoglobin was quenched with changes to the microenvironment of tyrosine and tryptophan residues and complex conformational changes of hemoglobin were induced with the loosening and unfolding skeleton. However, the heme in hemoglobin was still stable, indicating that the main physiological function of hemoglobin might not be significantly inhibited. This study will provide a new strategy to study the biological toxicity of QDs at the molecular level.     

Vitronectin (Vn) glycosylation patterned by lectin affinity assays—A potent glycoproteomic tool to discriminate plasma Vn from cancer ascites Vn

Changes in glycosylation have been associated with human cancer, but their complexity poses an analytical challenge. Ovarian cancer is a major cause of death in women because of an often late diagnosis. At least one‐third of patients presents ascites fluid at diagnosis, and almost all have ascites at recurrence. Vitronectin (Vn) is a multifunctional glycoprotein that is suggested to be implicated in ovarian cancer metastasis and is found within ascites. The present study evaluated the potential of using lectin affinity for characterizing the glycosylation pattern of Vn. Human Vn was purified from 1 sample of ovarian cancer ascites or a pool of plasma samples. Consistent findings were observed with both dot blot and lectin array assays. Based on a panel of 40 lectins, the lectin array revealed discriminant patterns of lectin binding to Vn glycans. Interestingly, almost all the highlighted interactions were found to be higher with Vn from ascites relative to the plasma counterpart. Also, the lectin array was able to discriminate profiles of lectin interactions (ConA, SNA‐I, PHA‐E, PHA‐L) between Vn samples that were not evident using dot blot, indicating its high sensitivity. The model of ConA binding during thermal unfolding of Vn confirmed the higher accessibility of mannosylated glycans in Vn from ascites as monitored by turbidimetry. Thus, this study demonstrated the usefulness of lectins and the lectin array as a glycoproteomic tool for high throughput and sensitive analysis of glycosylation patterns. Our data provide novel insights concerning Vn glycosylation patterns in clinical specimens, paving the way for further investigations regarding their functional impact and clinical interest.     

A calorimetric comparison of the interaction of sodium dodecyl sulfate with cytochrome c and erythrocyte glycoproteins.

The interactions of sodium dodecyl sulfate with cytochrome c and erythrocyte glycoproteins have been studied by the method of titration calorimetry. It was found that the initial addition of sodium dodecyl sulfate to cytochrome c caused an endothermic unfolding of the protein, detectable by circular dichroism (CD). This was followed by the exothermic binding of sodium dodecyl sulfate to the protein, without further CD-detectable conformational changes. In contrast, sodium dodecyl sulfate bound directly to the erythrocyte glycoproteins in an exothermic reaction without any accompanying CD-detectable conformation changes. This indicates that the glycoproteins solubilized in aqueous media have exposed hydrophobic regions which can interact directly with this detergent. The enthalpy changes and stoichiometries of binding are reported.     

Attributes of glycosylation in the establishment of the unfolding pathway of soybean agglutinin

Soybean agglutinin (gSBA) is a tetrameric legume lectin, each of whose subunits are glycosylated. Earlier studies have shown that this protein shows exceptionally high stability in terms of free energy of unfolding when compared to other proteins from the same family. This article deals with the unfolding reactions of the nonglycosylated recombinant form of the protein rSBA and its comparison with the glycosylated counterpart gSBA. The nonglycosylated form features a lower stability when compared to the glycosylated form. Further, the unfolding pathways in the two are widely different. Although the glycosylated form undergoes a simple two-state unfolding, the nonglycosylated species unfolds via a compact monomeric intermediate that is not a molten globule. Representative isothermal and thermal denaturation profiles show that glycosylation accounts for a stabilization of approximately 9 kcal/mol of the tetramer, whereas the difference in T(m) between the two forms is 26 degrees C. Computational studies on the glycan-protein interactions at the noncanonical interface of the protein show that quite a number of hydrogen bond and hydrophobic interactions stabilize the glycoprotein tetramer.     

Monitoring protein aggregation during thermal unfolding in circular dichroism experiments

Thermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heat-induced protein misfolding and aggregation often lead to formation of amyloid-like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/ unfolding transition by recording turbidity or 90 degrees light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 90 degrees light scattering can be done simultaneously with far- or near-UV CD data collection, they require no additional time or sample and can be directly correlated with the protein conformational changes monitored by CD. The results can provide useful insights into the origins of irreversible conformational changes and test the linkage between protein unfolding or misfolding and aggregation in various macromolecular systems, including globular proteins and protein-lipid complexes described in this study, as well as a wide range of amyloid-forming proteins and peptides.     

Intrinsic tryptophan fluorescence measurements suggest that polylactosaminyl glycosylation affects the protein conformation of the gelatin-binding domain from human placental fibronectin

Glycosylation can affect the physical and biochemical properties of the polypeptide chain in glycoproteins. Asparagine-N-linked polylactosaminyl glycosylation of the chymotryptic 44-kDa gelatin-binding domain from human placental fibronectin confers protease resistance [Zhu, B. C. R., Fisher, S. F., Panda, H., Calaycay, J., Shively, J. E. & Laine, R. A. (1984) J. Biol. Chem. 259, 3962-3970] and weaken the binding to gelatin [Zhu, B. C. R. & Laine, R. A. (1985) J. Biol. Chem. 260, 4041-4045]. Intrinsic tryptophan fluorescence of the gelatin-binding domain was used to probe glycosylation-dependent protein conformation changes. In gelatin-binding fragments containing incrementally smaller polylactosamine oligosaccharides, the fluorescence intensity progressively decreased and the emission spectrum shifted about 7 nm to the blue. Removal of the polylactosamine chains from a highly glycosylated fragment with endo-beta-galactosidase from Escherichia freundii also quenched the protein fluorescence. The fluorescence lifetimes did not appear to be affected by the extent of glycosylation, suggesting static quenching of the tryptophan emission in the low glycosylated fragments. Acrylamide quenching studies showed that the accessibility of the tryptophans to small solutes was not altered by glycosylation. The steady-state emission anisotropy increased with decreasing polylactosamine chain length. The results indicate that the polylactosamine chains alter the tryptophan environments in the gelatin-binding domain, probably by changing the polypeptide conformation. These putative protein conformation changes may be partially responsible for the altered gelatin binding, protease resistance, and cell adhesion functions of fetal tissue fibronectin.     

Unfolding studies on soybean agglutinin and concanavalin a tetramers: a comparative account

The unfolding pathway of two very similar tetrameric legume lectins soybean agglutinin (SBA) and Concanavalin A (ConA) were determined using GdnCl-induced denaturation. Both proteins displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provided values for conformational stability of the two proteins. It was found that the DeltaG of unfolding of SBA was much higher than ConA at all the temperatures at which the experiments were done. ConA had a T(g) 18 degrees C less than SBA. The higher conformational stability of SBA in comparison to ConA is largely due to substantial differences in their degrees of subunit interactions. Ionic interactions at the interface of the two proteins especially at the noncanonical interface seem to play a significant role in the observed stability differences between these two proteins. Furthermore, SBA is a glycoprotein with a GlcNac2Man9 chain attached to Asn-75 of each subunit. The sugar chain in SBA lies at the noncanonical interface of the protein, and it is found to interact with the amino acid residues in the adjacent noncanonical interface. These interactions further stabilize SBA with respect to ConA, which is not glycosylated.     

Interaction of locust apolipophorin III with lipoproteins and phospholipid vesicles: effect of glycosylation

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that binds reversibly to lipoprotein surfaces. The native protein is glycosylated at Asn-18 and Asn-85. Variable attachment of five distinct oligosaccharide moieties at the two glycosylation sites results in molecular weight heterogeneity, as seen by mass spectrometry. The main mass peak of 20,488 Da decreases to 17,583 Da after removal of carbohydrate, indicating that apoLp-III carbohydrate mass is approximately 14% by weight. Deglycosylated apoLp-III induced clearance of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol vesicles at a faster rate than glycosylated apoLp-III. However, in lipoprotein binding assays, in which apoLp-III interacts with surface-localized diacylglycerol, only minor differences in binding were observed. The fluorescence properties of 1-anilinonaphthalene-8-sulfonate were unaffected by the glycosylation state of apoLp-III, indicating that no changes in the relative amount of exposed hydrophobic surface occurred as a result of carbohydrate removal. We propose that glycosyl moieties affect the ability of apoLp-III to transform phospholipid bilayer vesicles into disc-like complexes by steric hindrance. This is due to the requirement that apoLp-III penetrate the bilayer substrate prior to conformational opening of the helix bundle. On the other hand, the glycosyl moieties do not affect lipoprotein binding interactions as it does not involve deep protein penetration into the lipid milieu. Rather, lipoprotein binding is based on oriented protein contact with the lipid surface followed by opening of the helix bundle, which allows formation of a stable interaction with surface exposed hydrophobic sites.     

Binding of pentagalloyl glucose to two globular proteins occurs via multiple surface sites

The interaction between pentagalloyl glucose (PGG) and two globular proteins, bovine serum albumin (BSA) and ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco), was investigated by isothermal titration calorimetry (ITC). ITC data fit to a binding model consisting of two sets of multiple binding sites, which reveal similarities in the mode of binding of PGG to BSA and rubisco. In both cases, the interaction is characterized by a high number of binding sites, which suggests that binding occurs by a surface adsorption mechanism that leads to coating of the protein surface, which promotes aggregation and precipitation of the PGG-protein complex. This model was confirmed by turbidimetry analysis of the PGG-BSA interaction. Analysis of tryptophan fluorescence quenching during the interaction of PGG with BSA suggests that binding of PGG leads to some conformational changes that are energetically closer to the unfolded state of the BSA structure, because small red shifts in the resulting emission spectra were observed.     

Amyloid fibrils formation and amorphous aggregation in concanavalin A

We here report an experimental study on the thermal aggregation process of concanavalin A, a protein belonging to the legume lectins family. The aggregation process and the involved conformational changes of the protein molecules were followed by means of fluorescence techniques, light scattering, circular dichroism, zeta potential measurements and atomic force microscopy. Our results show that the aggregation process of concanavalin A may evolve through two distinct pathways leading, respectively, to the formation of amyloids or amorphous aggregates. The relative extent of the two pathways is determined by pH, as amyloid aggregation is favored at high pH values ( approximately 9), while the formation of amorphous aggregates is favored at low pH ( approximately 5). At difference from amorphous aggregation, the formation of amyloid fibrils requires significant conformational changes on the protein, both at secondary and tertiary structural level. To our knowledge, this is the first observation of amyloid fibrils from concanavalin A.     

Non-glycosylated recombinant pro-concanavalin A is active without polypeptide cleavage.

The complex post-translational processing of concanavalin A (Con A) in maturing jackbeans is unique because the non-glycosylated mature active protein is circularly permuted in primary sequence relative to its own inactive precursor (glycosylated pro-Con A) and to other legume lectins. We show here that non-glycosylated pro-Con A expressed in bacteria from recombinant cDNA (rec-pro-Con A) folds in vivo and in vitro to a stable form which is active without further processing. N-glycosylation alone must therefore be sufficient to inactivate pro-Con A--a novel role for glycosylation in regulating activity during protein maturation.     

Calorimetry for studying the adsorption of proteins in hydrophobic interaction chromatography

Hydrophobic interaction chromatography is a very popular chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this frequent use, the complex interaction mechanism and the thermodynamic aspects of adsorption in hydrophobic interaction chromatography are still not well understood. Calorimetric methods such as isothermal titration calorimetry and flow calorimetry can help to gain a deeper understanding of the adsorption strength, the influence of salt type and temperature. They can be used to study conformational changes of proteins, which are often associated with the adsorption in hydrophobic interaction chromatography. This review offers a detailed introduction into the thermodynamic fundamentals of adsorption in hydrophobic interaction chromatography with a special focus on the potential applications of isothermal titration calorimetry and flow calorimetry for studying specific problems and relationships of the adsorption behavior of proteins and its various influencing factors. Models for characterizing conformational changes upon adsorption are presented together with methods for assessing this problem for different proteins and stationary phases. All of this knowledge can contribute greatly to forming a sound basis for method development, process optimization and finding modelling strategies in hydrophobic interaction chromatography.     

1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain

The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0–7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.     

Role of N-linked glycan in the unfolding pathway of Erythrina corallodendron lectin

Erythrina corallodendron lectin (ECorL) exhibits an exquisitely structured oligosaccharide chain. Interestingly, the bacterially expressed, nonglycosylated counterpart, rECorL, possesses an essentially identical carbohydrate specificity and agglutinating activity as the glycosylated lectin, thus suggesting that the overall structure of the two are identical. This paper reports the unfolding behavior of E. corallodendron lectin in its glycosylated (EcorL) and nonglycosylated (rECorL) forms. ECorL shows a two-state unfolding pattern during isothermal melts and differential scanning calorimetry (DSC). The T(g) of ECorL as obtained from isothermal melts is 74 degrees C at pH 7.4. The T(p) obtained from DSC studies is between 74.8 to 68.1 degrees C in the pH range of 5.26-7.77. The recombinant lectin (rECorL), which is devoid of carbohydrate, shows, in contrast to the glycosylated protein, a non-two-state unfolding profile as measured by both probes mentioned, but the number of intermediates during unfolding could not be ascertained. Simulated annealing on ECorL, with the sugars removed, reveals that the protein Calpha backbones overlap, indicating that the overall structure, including the mode of dimerization, of rECorL is insignificantly altered as compared to ECorL. The alterations in the folding behavior of rECorL as compared to that observed in ECorL may be due to the fact that, unlike most other glycoproteins, one of the glycans in ECorL is unusually structured and forms many hydrogen bonds with the protein. It therefore appears that while the covalently linked sugar does not contribute appreciably to the final folded structure of ECorL, it does alter its folding process in a significant manner.     

Molecular isoform distribution and glycosylation of acetylcholinesterase are altered in brain and cerebrospinal fluid of patients with Alzheimer's disease

The glycosylation of acetylcholinesterase (AChE) in CSF was analyzed by lectin binding. AChE from Alzheimer's disease (AD) patients was found to bind differently to two lectins, concanavalin A and wheat germ agglutinin, than AChE from controls. As multiple isoforms of AChE are present in both CSF and brain, we examined whether the abnormal glycosylation of AD AChE was due to changes in a specific molecular isoform. Globular amphiphilic dimeric (G2a) and monomeric (G1a) isoforms of AChE were found to be differentially glycosylated in AD CSF. Glycosylation of AChE was also altered in AD frontal cortex but not in cerebellum and was also associated with an increase in the proportion of light (G2 and G1) isoforms. This study demonstrates that the glycosylation of AChE is altered in the AD brain and that changes in AChE glycosylation in AD CSF may reflect changes in the distribution of brain isoforms. The study also suggests that glycosylation of AChE may be a useful diagnostic marker for AD.     

How does dextran sulfate prevent heat induced aggregation of protein? The mechanism and its limitation as aggregation inhibitor

The effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.1 and pH 6.2, while it had no effect at pH 7.5. Isothermal titration calorimetric analysis suggested that the pH dependency of the role of dextran sulfate on BSA aggregation would be related to the difference in the mode of BSA-dextran sulfate complex formation. Isothermal titration calorimetric analysis at pH 6.2 indicated that dextran sulfate did not bind to native BSA at this pH, but interacted with partially unfolded BSA. While stabilizing native form of protein by the complex formation has been suggested as the suitable mechanism of preventing aggregation, our observation of conformational changes by circular dichroism spectroscopy showed that strong electrostatic interaction between dextran sulfate and BSA rather facilitated the denaturation of BSA. Combining the data from isothermal titration calorimetry, circular dichroism, and dynamic light scattering, we found that the complex formation of the intermediate state of denatured BSA with dextran sulfate is a prerequisite to suppress the aggregation by preventing further oligomerization/aggregation process of denatured protein.     

Kinetically trapped metastable intermediate of a disulfide‐deficient mutant of the starch‐binding domain of glucoamylase

Refolding of a thermally unfolded disulfide‐deficient mutant of the starch‐binding domain of glucoamylase was investigated using differential scanning calorimetry, isothermal titration calorimetry, CD, and ¹H NMR. When the protein solution was rapidly cooled from a higher temperature, a kinetic intermediate was formed during refolding. The intermediate was unexpectedly stable compared with typical folding intermediates that have short half‐lives. It was shown that this intermediate contained substantial secondary structure and tertiary packing and had the same binding ability with β‐cyclodextrin as the native state, suggesting that the intermediate is highly‐ordered and native‐like on the whole. These characteristics differ from those of partially folded intermediates such as molten globule states. Far‐UV CD spectra showed that the secondary structure was once disrupted during the transition from the intermediate to the native state. These results suggest that the intermediate could be an off‐pathway type, possibly a misfolded state, that has to undergo unfolding on its way to the native state.     

Unfolding transitions of fibronectin and its domains. Stabilization and structural alteration of the N-terminal domain by heparin.

Changes in the conformational state of human plasma fibronectin and several of its fragments were studied by fluorescence emission, intrinsic fluorescence polarization and c.d. spectroscopy under conditions of guanidinium chloride-and temperature-induced unfolding. Fragments were chosen to represent all three types of internal structural homology in the protein. Low concentration (less than 2 M) of guanidinium chloride induced a gradual transition in the intact protein that was not characteristic of any of the isolated domains, suggesting the presence of interdomain interactions within the protein. Intermediate concentrations of guanidinium chloride (2-3 M) and moderately elevated temperatures (55-60 degrees C) induced a highly co-operative structural transition in intact fibronectin that was attributable to the central 110 kDa cell-binding domain. High temperatures (greater than 60 degrees C) produced a gradual unfolding in the intact protein attributable to the 29 kDa N-terminal heparin-binding and 40 kDa collagen-binding domains. Binding of heparin to intact fibronectin and to its N-terminal fragment stabilized the proteins against thermal unfolding. This was reflected in increased delta H for the unfolding transitions of the heparin-bound N-terminal fragment, as well as decreased accessibility to solvent perturbants of internal chromophores in this fragment when bound to heparin. These results help to account for the biological efficacy of the interaction between the fibronectin N-terminal domain and heparin, despite its relatively low affinity.     

Electrostatic selectivity in protein-nanoparticle interactions

The binding of bovine serum albumin (BSA) and β-lactoglobulin (BLG) to TTMA (a cationic gold nanoparticle coupled to 3,6,9,12-tetraoxatricosan-1-aminium, 23-mercapto-N,N,N-trimethyl) was studied by high-resolution turbidimetry (to observe a critical pH for binding), dynamic light scattering (to monitor particle growth), and isothermal titration calorimetry (to measure binding energetics), all as a function of pH and ionic strength. Distinctively higher affinities observed for BLG versus BSA, despite the lower pI of the latter, were explained in terms of their different charge anisotropies, namely, the negative charge patch of BLG. To confirm this effect, we studied two isoforms of BLG that differ in only two amino acids. Significantly stronger binding to BLGA could be attributed to the presence of the additional aspartates in the negative charge domain for the BLG dimer, best portrayed in DelPhi. This selectivity decreases at low ionic strength, at which both isoforms bind well below pI. Selectivity increases with ionic strength for BLG versus BSA, which binds above pI. This result points to the diminished role of long-range repulsions for binding above pI. Dynamic light scattering reveals a tendency for higher-order aggregation for TTMA-BSA at pH above the pI of BSA, due to its ability to bridge nanoparticles. In contrast, soluble BLG-TTMA complexes were stable over a range of pH because the charge anisotropy of this protein at makes it unable to bridge nanoparticles. Finally, isothermal titration calorimetry shows endoenthalpic binding for all proteins: the higher affinity of TTMA for BLGA versus BLGB comes from a difference in the dominant entropy term.