bis-ANS Fluorescence Research Articles

Tetramerization of the S100B Chaperone Spawns a Ca2+ Independent Regulatory Surface that Enhances Anti-aggregation Activity and Client Specificity

Alzheimer’s disease (AD) hallmarks include the aggregation of amyloid-β (Aβ), tau and neuroinflammation promoted by several alarmins. Among these is S100B, a small astrocytic homodimeric protein, upregulated in AD, whose multiple biological activities depend on localization, concentration, and assembly state. S100B was reported to inhibit the aggregation and toxicity of Aβ42 and tau similarly to a holdase-type chaperone. This activity is dependent of Ca2+-binding, which triggers the exposure of a regulatory binding cleft at the S100B dimer interface with which amyloidogenic clients dynamically interact. Although the dimer prevails, a significant portion of secreted S100B in the human brain occurs as higher order multimers, whose protective functions remain uncharacterized and which we here investigate. Resorting to ThT-monitored aggregation kinetics, we determined that unlike the dimer, tetrameric S100B inhibits Aβ42 aggregation at sub/equimolar ratios, an effect that persists in the absence of Ca2+ binding. Structural analysis revealed that S100B tetramerization spawns a novel extended cleft accommodating an aggregation-prone surface that mediates interactions with monomeric Aβ client via hydrophobic interactions, as corroborated by Bis-ANS fluorescence and docking analysis. Correspondingly, at high ionic strength that reduces solvation and favours hydrophobic contacts, the inhibition of Aβ42 aggregation by tetrameric S100B is 3-fold increased. Interestingly, this extended Ca2+-independent surface favours Aβ42 as substrate, as tau K18 aggregation is not inhibited by the apo tetramer. Overall, results illustrate a mechanism through which oligomerization of the S100B chaperone fine-tunes anti-aggregation activity and client specificity, highlighting the potential functional relevance of S100B multimers in the regulation of AD proteotoxicity.

P53 TAD2 Domain (38-61) Forms Amyloid-like Aggregates in Isolation.

A strong association between protein aggregation and human diseases (such as Alzheimer's, Parkinson's, and Huntington's disease) is well demonstrated. Misfolding and aggregation of p53, a central transcriptional mediator, has been revealed by various experimental evidence in different types of cancers. Aggregation studies focusing on different p53 domains, mostly, the central core domain and its mutants under the influence of various environmental conditions, and the p53 transactivation domain (TAD) (1-63) have been reported. However, the specific subdomains responsible for p53 aggregation are not known. p53 TADs interact with diverse cellular factors to modulate the function of p53 and elicit appropriate cellular responses under different stress conditions. In this study, the aggregation of the p53 TAD2 domain (38-61) has been studied in isolation. The aggregates were generated in vitro under acidic pH conditions after in silico scoring for amyloidogenic tendency and characterized using dye-based assays (ThT and bis-ANS fluorescence), CD spectroscopy, and microscopy (scanning electron microscoy, transmission electron microscopy, and atomic force microscopy). It was observed that p53 TAD2 forms characteristic β-sheet-rich amyloid-like fibrils. Via a reductionist approach, this study highlights the nature of p53 TAD2 domain (38-61) aggregation.

Hydrogel Coatings on Container Surfaces Reduce Protein Aggregation Caused by Mechanical Stress and Cavitation.

This work reports the ability of hydrogel coatings to protect therapeutic proteins from cavitation-induced aggregation caused by mechanical stress. Here, we show that polyacrylamide hydrogel coatings on container surfaces suppress mechanical shock-induced cavitation and the associated aggregation of intravenous immunoglobulin (IVIg). First, crosslinked polyacrylamide hydrogels were grown on the surfaces of borosilicate glass vials. Treatment with ultrasound showed that these hydrogel surfaces suppressed cavitation events to levels below those found for unfunctionalized borosilicate glass. Next, IVIg solutions were loaded into these vials and subjected to tumbling, horizontal shaking, and drop testing. Aggregation was quantified by bisANS fluorescence staining and particle counting by flow imaging microscopy (FIM). In all cases, the presence of polyacrylamide hydrogels on the vial surfaces reduced the amount of IVIg aggregation and the number of particulates. In addition, the polyacrylamide appeared to have a protective effect that prevented additional aggregates from forming at extended tumbling times. Finally, drop test studies showed that the polyacrylamide coatings suppressed detectable cavitation. This work reveals how even a simple hydrogel vial coating can have a profound effect on stabilizing protein therapeutics.

Isomerization of Asp is essential for assembly of amyloid-like fibrils of αA-crystallin-derived peptide.

Post-translational modifications are often detected in age-related diseases associated with protein misfolding such as cataracts from aged lenses. One of the major post-translational modifications is the isomerization of aspartate residues (L-isoAsp), which could be non-enzymatically and spontaneously occurring in proteins, resulting in various effects on the structure and function of proteins including short peptides. We have reported that the structure and function of an αA66–80 peptide, corresponding to the 66–80 (66SDRDKFVIFLDVKHF80) fragment of human lens αA-crystallin, was dramatically altered by the isomerization of aspartate residue (Asp) at position 76. In the current study, we observed amyloid-like fibrils of L-isoAsp containing αA66–80 using electron microscopy. The contribution of each amino acid for the peptide structure was further evaluated by circular dichroism (CD), bis-ANS, and thioflavin T fluorescence using 14 alanine substituents of αA66–80, including L-isoAsp at position 76. CD of 14 alanine substituents demonstrated random coiled structures except for the substituents of positively charged residues. Bis-ANS fluorescence of peptide with substitution of hydrophobic residue with alanine revealed decreased hydrophobicity of the peptide. Thioflavin T fluorescence also showed that the hydrophobicity around Asp76 of the peptide is important for the formation of amyloid-like fibrils. One of the substitutes, H79A (SDRDKFVIFL(L-isoD)VKAF) demonstrated an exact β-sheet structure in CD and highly increased Thioflavin T fluorescence. This phenomenon was inhibited by the addition of protein-L-isoaspartate O-methyltransferase (PIMT), which is an enzyme that changes L-isoAsp into Asp. These interactions were observed even after the formation of amyloid-like fibrils. Thus, isomerization of Asp in peptide is key to form fibrils of αA-crystallin-derived peptide, and L-isoAsp on fibrils can be a candidate for disassembling amyloid-like fibrils of αA-crystallin-derived peptides.

Apolipoprotein E4 exhibits intermediates with domain interaction

ApoE4(C112R) is the strongest risk factor for Alzheimer's disease, while apoE3(C112) is considered normal. The C112R substitution is believed to alter the interactions between the N-terminal (NTD) and the C-terminal domain (CTD) leading to major functional differences. Here we investigate how the molecular property of the residue at position 112 affects domain interaction using an array of C112X substitutions with arginine, alanine, threonine, valine, leucine and isoleucine as ‘X’. We attempt to determine the free energy of domain interaction (∆GINT) from stabilities of the NTD (∆GNTD) and CTD (∆GCTD) in the full-length apoE, and the stabilities of fragments of the NTD (∆GNTF) and CTD (∆GCTF), using the relationship, ∆GINT = ∆GNTD + ∆GCTD - ∆GNTF - ∆GCTF. We find that although ∆GNTD is strongly dependent on the C112X substitutions, ∆GNTD - ∆GNTF is small. Furthermore, ∆GCTD remains nearly the same as ∆GCTF. Therefore, ∆GINT is estimated to be small and similar for the apoE isoforms. However, stability of domain interaction monitored by urea dependent changes in interdomain Forster Resonance Energy Transfer (FRET) is found to be strongly dependent on C112X substitutions. ApoE4 exhibits the highest mid-point of denaturation of interdomain FRET. To resolve the apparently contradictory observations, we hypothesize that higher interdomain FRET in apoE4 in urea may involve ‘intermediate’ states. Enhanced fluorescence of bis-ANS and susceptibility to proteolytic cleavage support that apoE4, specifically, the NTD of apoE4 harbor ‘intermediates’ in both native and mildly denaturing conditions. The intermediates could hold key to the pathological functions of apoE4.

Effect of Cu2+ and Zn2+ ions on human serum albumin interaction with plasma unsaturated fatty acids.

Human serum albumin (HSA) serves as a depot and carrier of multiple unrelated ligands including several participants of the pathogenesis of Alzheimer's disease (AD), such as amyloid β peptide (Aβ), Zn2+/Cu2+ ions, docosahexaenoic (DHA), linoleic (LA), and oleic (OA) acids. To explore the interplay between HSA interaction with Zn2+/Cu2+ and the plasma unsaturated fatty acids (DHA, LA, OA, and arachidonic acid (ArA)), we have studied the metal dependence of the fatty acid (FA) binding capacity of HSA (nmax) and structural consequences of the HSA-FA interactions. HSA loading with Zn2+ decreases nmax value by 0.3-1.5, while its saturation with Cu2+ causes the FA-dependent nmax changes by up to 0.9. The Cu2+-induced decline in nmax value for DHA is due to conformational rearrangements in HSA molecule. In other cases, the changes in nmax are attributed to steric hindarance/facilitation of the HSA-FA interaction because of the protein multimerization/monomerization, as confirmed by chemical crosslinking. The surface hydrophobicity of HSA is Cu2+-, Zn2+-, and FA-dependent and decreases upon the FA binding, according to bis-ANS fluorescence data. Overall, Zn2+ or Cu2+ binding selectively affect HSA interaction with the FAs studied, in part due to changes in quaternary structure of the protein.

The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin.

αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core "α-crystallin" domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54-60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54-60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54-60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54-60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone.

The role of resveratrol and melatonin in the nitric oxide and its oxidation products mediated functional and structural modifications of two glycolytic enzymes: GAPDH and LDH

BackgroundNitric oxide is a well-known gaseous signaling molecule and protein modifying agent. However, at higher concentrations or during oxidative stress nitric oxide may exert some deleterious effects on protein structure and function. Here we investigated the influence of nitric oxide and products of its oxidation on two glycolytic enzymes: GAPDH and LDH under in vitro nitrosative stress conditions. Secondly, we applied natural antioxidants: melatonin and resveratrol to examine their effects on the enzymes under studied conditions. MethodsBy means of UV–VIS and fluorescence spectroscopy methods we compared nitric oxide mediated changes of enzyme activities, amount of free sulfhydryl groups (-SH) and bis-ANS probe binding. Finally, we predicted potential cysteine residues modified by nitric oxide in studied proteins using GPS-SNO software. ResultsOur results indicated that nitric oxide reversibly inactivates GAPDH but does not affect the activity of LDH. Nitric oxide dependent GAPDH activity decline was accompanied by the reduction of the amount of free –SH groups and GAPDH-bound bis-ANS fluorescence. Reduction of the number of free –SH groups and protein-bound bis-ANS fluorescence was also observed in LDH treated with NO. Applied antioxidants increased inactivation of GAPDH and structural changes of GAPDH and LDH. ConclusionsNitric oxide modifies function and structure of thiol-dependent enzyme such as GAPDH and structure of LDH which function do not rely on cysteine thiols. Both resveratrol and melatonin exerted prooxidative properties in studied conditions. General significanceExtensively studied antioxidants: resveratrol and melatonin may function as a prooxidative species under in vitro nitrosative stress conditions.

Strong Inhibitory Effect of Heme on hIAPP Fibrillation.

The deposition of human islet amyloid polypeptide (hIAPP) within β-cells is implicated in the etiology of type 2 diabetes mellitus (T2Dm). It was reported that heme could bind to hIAPP. We speculate that binding may affect the aggregation of hIAPP. In this study, UV-vis spectroscopy was used to detect the interaction pattern between the heme and hIAPP. ThT and Bis-ANS fluorescence assay, circular dichroism spectroscopy, gel electrophoresis assay, and transmission electron microscopy were employed to study the effect of heme on the aggregation of hIAPP. We found that heme dramatically inhibited hIAPP aggregation, even partially dismantled hIAPP aggregates by preventing its conformational changes. Moreover, a similar inhibitory effect was also observed on mutant hIAPP. In the compared group, the inhibitory effects of protoporphyrin on hIAPP and its mutants aggregation were weaker. Similarly, its effect on the dismantlement of the aggregates was also weaker. On the basis of these results, we revealed that the heme iron center was not required for the inhibitory effect on hIAPP but affected the binding affinity of heme to hIAPP. Besides Arg11 and His18, other hydrophobic residues of hIAPP may also play important roles in heme binding. Our results may help to develop an in-depth understanding of the interaction between heme and hIAPP, which would be helpful in designing new therapeutic strategies against T2Dm.

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies

Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.

Disulfide-bond scrambling promotes amorphous aggregates in lysozyme and bovine serum albumin.

Disulfide bonds are naturally formed in more than 50% of amyloidogenic proteins, but the exact role of disulfide bonds in protein aggregation is still not well-understood. The intracellular reducing agents and/or improper use of antioxidants in extracellular environment can break proteins disulfide bonds, making them unstable and prone to misfolding and aggregation. In this study, we report the effect of disulfide-reducing agent dithiothreitol (DTT) on hen egg white lysozyme (lysozyme) and bovine serum albumin (BSA) aggregation at pH 7.2 and 37 °C. BSA and lysozyme proteins treated with disulfide-reducing agents form very distinct amorphous aggregates as observed by scanning electron microscope. However, proteins with intact disulfide bonds were stable and did not aggregate over time. BSA and lysozyme aggregates show unique but measurable differences in 8-anilino-1-naphthalenesulfonic acid (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, suggesting a loose and flexible aggregate structure for lysozyme but a more compact aggregate structure for BSA. Scrambled disulfide-bonded protein aggregates were observed by nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for both proteins. Similar amorphous aggregates were also generated using a nonthiol-based reducing agent, tris(2-carboxyethyl)phosphine (TCEP), at pH 7.2 and 37 °C. In summary, formation of distinct amorphous aggregates by disulfide-reduced BSA and lysozyme suggests an alternate pathway for protein aggregation that may be relevant to several proteins.

Real-time heterogeneous protein-protein interaction between αA-crystallin N-terminal mutants and αB-crystallin using quartz crystal microbalance (QCM).

The lens transparency depends on higher concentration of lens proteins and their interactions. α-Crystallin is one of the predominant lens proteins, responsible for proper structural and functional architecture of the lens microenvironment, and any alteration of which results in cataract formation. The R12C, R21L, R49C and R54C are the most significant and prevalent αA-crystallin congenital cataract-causing mutants worldwide. Protein-protein interaction, crucial for lens proper structure and function, was posited to be lost due to point mutation and the elucidation of which could shed light on the molecular basis of cataract. In this conjuncture, we report quartz crystal microbalance (QCM) as a warranted technique for real-time analysis of protein-protein interaction between the N-terminal mutants of αA-crystallin and αB-crystallin. The biophysical characteristics of the mutated proteins were determined by size-exclusion HPLC, far-UV circular dichroism and fluorescence studies. Far-UV circular dichroism spectral analysis displayed slight modifications in β-sheet of R54C mutant. Altered intrinsic tryptophan fluorescence and decreased bis-ANS fluorescence were observed in all the N-terminal mutations revealing the tertiary structural changes and decreased exposure of surface hydrophobicity. An emphatic fall in the chaperone activity was observed in the N-terminal mutants, R12C, R21L and R54C. QCM analysis revealed the occurrence of strong heterogeneous interaction between αA-crystallin and αB-crystallin. Nevertheless, decreased interactions were observed with the N-terminal mutants. In summary, the present study concludes that the loss of interactions between αA-crystallin N-terminal mutants and αB-crystallin signifies quaternary structural alterations due to mutation in the arginine residues.

Structural Behavior of Cardiac Troponin C Variants Present in Cardiomyopathic Patients

Cardiac Troponin C (TnC) is a Ca2+-binding protein and plays an important role in regulation of muscle contraction. Mutations in cTnC are implicated in phenotypic characteristics known as hypertrophic and dilated cardiomyopathy (HCM and DCM, respectively). However, the structural mechanisms underlying cardiac dysfunction are unknown. The main goal of this work is to investigate changes in stability and dynamics of seven cTnC variants (A8V, D145E, C84Y and A31S related to HCM; and Y5H, M103I and I148V related to DCM) using an ensemble of thermodynamic and structural approaches. Ca2+-titrations monitored by bis-ANS fluorescence revealed that D145E, A31S and all mutations related with DCM decreased the Ca2+-induced hydrophobic exposure, while C84Y substantially enhance it by the N-domain exposure compared to WT. Thermostability monitored by circular dichroism revealed similar melting temperatures between apo and holo states for D145E (apo: 66.4 ± 1.4°C, holo: 65.4 ± 1.6°C) but different values for WT (apo: 65 ± 1.9°C and holo: >90°C) and C84Y (apo: 43.8 ± 1.5°C, holo: 66.6 ± 0.8°C). Shape restorations from small angle X-ray scattering were used to evaluate conformational changes induced by the mutations. In the apo state, an increase in the radius of gyration values upon increasing concentrations of urea was observed for the Y5H, A31S, M103I and I148V mutants compared to WT. Furthermore, the D145E displayed the most affected shape compared to WT and perturbed residues were located at the C-domain as confirmed by chemical shift perturbation analysis. In addition, the D145E secondary structure was not significantly altered by dihedral angles prediction from the NMR assignment data. These observations open up new avenues for the comprehension of the complex behavior of HCM and DCM mutations in cTnC that has heretofore been not evaluated at structural level.

Physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathies

Some physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathy were analyzed. Mutation K141Q did not affect intrinsic Trp fluorescence and interaction with hydrophobic probe bis-ANS, whereas mutation R140G decreased both intrinsic fluorescence and fluorescence of bis-ANS bound to HspB1. Both mutations decreased thermal stability of HspB1. Mutation R140G increased, whereas mutation K141Q decreased the rate of trypsinolysis of the central part (residues 5-188) of HspB1. Both the wild type HspB1 and its K141Q mutant formed large oligomers with apparent molecular weight ∼560kDa. The R140G mutant formed two types of oligomers, i.e. large oligomers tending to aggregate and small oligomers with apparent molecular weight ∼70kDa. The wild type HspB1 formed mixed homooligomers with R140G mutant with apparent molecular weight ∼610kDa. The R140G mutant was unable to form high molecular weight heterooligomers with HspB6, whereas the K141Q mutant formed two types of heterooligomers with HspB6. Invitro measured chaperone-like activity of the wild type HspB1 was comparable with that of K141Q mutant and was much higher than that of R140G mutant. Mutations of homologous hot-spot Arg (R140G of HspB1 and R120G of αB-crystallin) induced similar changes in the properties of two small heat shock proteins, whereas mutations of two neighboring residues (R140 and K141) induced different changes in the properties of HspB1.

Inactivation and fragmentation of lectin from Bothrops leucurus snake venom by gamma irradiation

Gamma radiation alters the molecular structure of biomolecules and is able to mitigate the action of snake venoms and their isolated toxins. The effect of γ-radiation on the folding of Bothrops lecurus venom lectin was measured by a hemagglutinating assay, intrinsic and bis-ANS fluorescence. Intrinsic and bis-ANS fluorescence analyses indicated that irradiation caused unfolding followed by aggregation of the lectin. Our results suggest that irradiation can lead to significant changes in the protein structure, which may promote the loss of its binding property and toxic action.

Identification of regions responsible for the open conformation of S100A10 using chimaeric S100A11–S100A10 proteins

S100A11 is a dimeric EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2) and facilitate membrane vesiculation events. In contrast with other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an 'open' conformation that is very similar to S100A11 in its calcium-bound state. To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimaeric proteins were constructed where regions from calcium-binding sites I and II, and helices II-IV in S100A11 were replaced with the corresponding regions of S100A10. The chimaeric proteins having substitutions in calcium-binding site II displayed increased hydrophobic surface exposure as assessed by bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'disulfonic acid, dipotassium salt) fluorescence and phenyl-Sepharose binding in the absence of calcium. This response is similar to that observed for Ca2+-S100A11 and calcium-free S100A10. Further, this substitution resulted in calcium-insensitive binding to annexin A2 for one chimaeric protein. The results indicate that residues within site II are important in stabilizing the open conformation of S100A10 and presentation of its target binding site. In contrast, S100A11 chimaeric proteins with helical substitutions displayed poorer hydrophobic surface exposure and, consequently, unobservable annexin A2 binding. The present study represents a first attempt to systematically understand the molecular basis for the calcium-insensitive open conformation of S100A10.

Comparison of analytical methods to detect instability of etanercept during thermal stress testing

The aim was comparing the capability of a set of analytical methods to detect physical instability (focus on aggregation and structural changes) of etanercept during thermal stress testing as early as possible. Pre-filled syringes of Enbrel® 50 mg from three batches were thermally stressed for one week at 50 °C. Samples were taken at days 0, 1, 2, 3, 4 and 7, and analyzed with high-performance liquid size exclusion chromatography (HP-SEC), SDS–PAGE gel electrophoresis, dynamic light scattering (DLS), light obscuration, extrinsic fluorescence (Bis-ANS), far-UV circular dichroism (CD) spectroscopy, second derivative UV spectroscopy (UV), and enzyme-linked immunosorbent assay (ELISA). Thermal stress resulted in the formation of small soluble aggregates (HP-SEC, DLS) which were in part covalent (SDS–PAGE), and conformationally changed (Bis-ANS, CD, UV). No significant increase in subvisible particles was detected by light obscuration. An apparent increase in TNF-α binding to etancercept in the stressed formulations was found by ELISA. The three batches were comparable when unstressed, but showed slight differences in aggregation tendency. Bis-ANS fluorescence was most sensitive with respect to early-stage detection of heat-induced instability of etanercept (significant changes already at day 1), followed by HP-SEC (day 2) and DLS (day 3). This points towards a degradation mechanism involving exposure of hydrophobic patches due to partial unfolding followed by aggregation.

Gamma irradiation as an alternative treatment to abolish allergenicity of lectins in food

Food irradiation is the process of exposing food to ionising radiation to eliminate microorganisms or insects. Lectins are the most common agents in food intolerance, and efficient methods to reduce unwanted or intolerant immunological effects of lectins have not yet been described. Sebastiania jacobinensis bark lectin was structurally altered after gamma irradiation. Hemagglutination assays showed that the lectin was stimulated by low doses of radiation (0.1 kGy), while high doses (above 1 kGy) induced a significant loss of activity. The effect of γ-radiation on lectin hydrophobicity was measured by intrinsic and bis-ANS fluorescence. High doses of ionising radiation suppressed the intrinsic fluorescence emission and promoted polypeptide fragmentation and hydrophobic surface modification. The results suggested that changes in the hydrophobic surface induced by gamma irradiation led to protein misfolding and, subsequently, to aggregation. The pioneering viewpoints presented on the stability of a food allergen after gamma irradiation might contribute to the development of harmless and more effective methods to reduce or eliminate food allergenicity.

Magnesium Stabilizes the Closed Conformation of the C-Domain of Troponin C

Activation of the thin filament in striated muscles requires both the binding of Ca2+ to the N-domain of troponin C (TnC) and the binding of myosin crossbridges to actin, which has been shown to alter the C-domain conformation. Here we have evaluated the structural and functional consequences of divalent cation exchange in skeletal and cardiac TnC (sTnC and cTnC). We have used intrinsic tyrosine fluorescence, circular dichrosim (CD), and the fluorescent nonspecific hydrophobic probe bis-ANS to monitor changes in domain conformation in response to Ca2+ and Mg2+ binding in the sTnC, cTnC, and in a cTnC mutant in which the invariant Glu residue at the 12th position of the calcium binding loops III and IV were substituted with Asp (cTnCDD). Ca2+ binding causes an increase in Tyr fluorescence and α-helical content in sTnC and cTnC, but not in cTnCDD. Ca2+ induced C-domain opening characteristic of sTnC and cTnC was also greatly reduced in cTnCDD, as measured by bis-ANS fluorescence. Thus the Asp to Glu substitutions appear to prevent the C-domain from opening. Bis-ANS Ca2+ titrations also showed that high Ca2+ concentrations may be sufficient to open the N-domain of cTnC, which was reported to remain in the closed conformation in the Ca2+-bound state. Lastly, bis-ANS Mg2+ titrations indicate that Mg2+ does not cause domain opening in either cTnC or cTnCDD. The closed conformation of the Mg2+-bound C-domain of TnC implies a different mechanism of interaction with TnI than that in the presence of Ca2+ and suggests that the Mg2+- Ca2+exchange in TnC may contribute to the thin filament activation of muscle contraction. This conclusion is consistent with our observation that physiological concentrations of Mg2+ significantly lower the Ca2+-sensitivity of reconstituted cardiac thin filaments.

Kinetic Instability of the Serpin Z α1-Antitrypsin Promotes Aggregation

The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is alpha(1)-antitrypsin (alpha(1)AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of alpha(1)AT, which accounts for 95% of all cases of alpha(1)AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z alpha(1)AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type alpha(1)AT and Z alpha(1)AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type alpha(1)AT and Z alpha(1)AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z alpha(1)AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed.