Tryptophan Fluorescence Spectroscopy Research Articles

Discovery of periplasmic solute binding proteins with specificity for ketone bodies: β-hydroxybutyrate binding proteins.

Polyhydroxyalkanoates are a class of biodegradable, thermoplastic polymers which represent a major carbon source for various bacteria. Proteins which mediate the translocation of polyhydroxyalkanoate breakdown products, such as β-hydroxybutyrate (BHB)-a ketone body which in humans serves as an important biomarker, have not been well characterized. In our investigation to screen a solute-binding protein (SBP) which can act as a suitable recognition element for BHB, we uncovered insights at the intersection of bacterial metabolism and diagnostics. Herein, we identify SBPs associated with putative ATP-binding cassette transporters that specifically recognize BHB, with the potential to serve as recognition elements for continuous quantification of this analyte. Through bioinformatic analysis, we identified candidate SBPs from known metabolizers of polyhydroxybutyrate-including proteins from Cupriavidus necator, Ensifer meliloti, Paucimonas lemoignei, and Thermus thermophilus. After recombinant expression in Escherichia coli, we demonstrated with intrinsic tryptophan fluorescence spectroscopy that four candidate proteins interacted with BHB, ranging from nanomolar to micromolar affinity. Tt.2, an intrinsically thermostable protein from Thermus thermophilus, was observed to have the tightest binding and specificity for BHB, which was confirmed by isothermal calorimetry. Structural analyses facilitated by AlphaFold2, along with molecular docking and dynamics simulations, were used to hypothesize key residues in the binding pocket and to model the conformational dynamics of substrate unbinding. Overall, this study provides strong evidence identifying the cognate ligands of SBPs which we hypothesize to be involved in prokaryotic cellular translocation of polyhydroxyalkanoate breakdown products, while highlighting these proteins' promising biotechnological application.

Intermolecular Interactions between Cysteine and Aromatic Amino Acids with a Phenyl Moiety in the DNA-Binding Domain of Heat Shock Factor 1 Regulate Thermal Stress-Induced Trimerization.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human (Homo sapiens), goldfish (Carassius auratus), and walleye pollock (Gadus chalcogrammus) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

Transfer of ANS-Like Drugs from Micellar Drug Delivery Systems to Albumin Is Highly Favorable and Protected from Competition with Surfactant by "Reserved" Binding Sites.

Micellar drug delivery systems (MDDS) for the intravenous administration of poorly soluble drugs have great advantages over alternative formulations in terms of the safety of their excipients, storage stability, and straightforward production. A classic example is mixed micelles of glycocholate (GC) and lecithin, both endogenous substances in human blood. What limits the use of MDDS is the complexity of the transitions after injection. In particular, as the MDDS disintegrate partially or completely after injection, the drug has to be transferred safely to endogenous carriers in the blood, such as human serum albumin (HSA). If this transfer is compromised, the drug might precipitate─a process that needs to be excluded under all circumstances. The key question of this paper is whether the high local concentration of GC at the moment and site of MDDS dissolution might transiently saturate HSA binding sites and, hence, endanger quick drug transfer. To address this question, we have used a new approach, which is time-resolved fluorescence spectroscopy of the single tryptophan in HSA, Trp-214, to characterize the competitive binding of GC and the drug substitute anilinonaphthalenesulfonate (ANS) to HSA. Time-resolved fluorescence of Trp-214 showed important advantages over established methods for tackling this problem. ANS has been the standard "model drug" to study albumin binding for decades, given its structural similarity to the class of naphthalene-containing acidic drugs and the fact that it is displaced from HSA by numerous drugs (which presumably bind to the same sites). Our complex global fit uses the critical approximation that the average lifetimes behave similarly to a single lifetime, but the resulting errors are found to be moderate and the results provide a convincing explanation of the, at first glance, counterintuitive behavior. Accordingly, and largely in line with the literature, we observed two types of sites binding ANS at HSA: 3 type A, rather peripheral, and 2 type B, likely more central sites. The latter quench Trp-214 by Förster Resonance Energy Transfer (FRET) with a rate constant of ≈0.4 ns-1 per ANS. Adding millimolar concentrations of GC displaces ANS from the A sites but not from B sites. At incomplete ANS saturation, this causes a GC-induced translocation of ANS from A to the more FRET-active B sites. This leads to the apparent paradox that the partial displacement of ANS from HSA increases its quenching effect on Trp-214. The most important conclusion is that (ANS-like) drugs cannot be displaced from the type-B sites, and consequently, drug transfer to these sites is not impaired by competitive binding of GC in the vicinity of a dissolving micelle. The second conclusion is that for unbound GC above the CMC (9 mM), ANS equilibrates between HSA and GC micelles but with a strong preference for free sites on HSA. That means that even persisting micelles would lose their cargo readily once exposed to HSA. For all MDDS sharing this property, targeted drug delivery approaches involving them as the nanocarrier would be pointless.

Polyethylene Glycol Impacts Conformation and Dynamics of Escherichia coli Prolyl-tRNA Synthetase Via Crowding and Confinement Effects.

Polyethylene glycol (PEG) is a flexible, nontoxic polymer commonly used in biological and medical research, and it is generally regarded as biologically inert. PEG molecules of variable sizes are also used as crowding agents to mimic intracellular environments. A recent study with PEG crowders revealed decreased catalytic activity of Escherichia coli prolyl-tRNA synthetase (Ec ProRS), where the smaller molecular weight PEGs had the maximum impact. The molecular mechanism of the crowding effects of PEGs is not clearly understood. PEG may impact protein conformation and dynamics, thus its function. In the present study, the effects of PEG molecules of various molecular weights and concentrations on the conformation and dynamics of Ec ProRS were investigated using a combined experimental and computational approach including intrinsic tryptophan fluorescence spectroscopy, atomic force microscopy, and atomistic molecular dynamic simulations. Results of the present study suggest that lower molecular weight PEGs in the dilute regime have modest effects on the conformational dynamics of Ec ProRS but impact the catalytic function primarily via the excluded volume effect; they form large clusters blocking the active site pocket. In contrast, the larger molecular weight PEGs in dilute to semidilute regimes have a significant impact on the protein's conformational dynamics; they wrap on the protein surface through noncovalent interactions. Thus, lower-molecular-weight PEG molecules impact protein dynamics and function via crowding effects, whereas larger PEGs induce confinement effects. These results have implications for the development of inhibitors for protein targets in a crowded cellular environment.

Amino acids and glycine derivatives differently affect refolding of mesophilic and thermophilic like α-amylases: implications in protein refolding and aggregation

α-amylases are industrially important enzymes which are used in different starch-based industries. They are adapted to different environmental conditions like extremes of temperature, pH and salinity. Herein, α-amylases from Bacillus amyloliquifaciens (BAA) and Bacillus licheniformis (BLA), representing mesophilic and thermophilic-like proteins, respectively, have been used to investigate the effect of naturally occurring osmolytes like arginine, proline, glycine and its methyl derivatives, sarcosine and betaine on their refolding. In this study, we have shown that among amino acids and glycine derivatives, betaine is the most promising osmolyte, while arginine and glycine exhibit moderately positive effect at their lower concentrations on the refolding of BAA only. Except betaine, all other osmolytes above 0.25 M showed inhibitory effect on the native enzyme activity of BLA and BAA. However, aggregation kinetics monitored by static light scattering indicates suppression of aggregation by all of these osmolytes. Further investigation by tryptophan and ANS fluorescence spectroscopy indicates the formation of compact hydrophobic core in the presence of the osmolytes. The morphology of protein aggregates having different sizes was visualized by atomic force microscopy ,and it was observed that amorphous aggregates of variable heights were formed. Our study highlights the importance of differential effects of arginine, proline, glycine, sarcosine and betaine on the native state as well as on refolding of BLA and BAA which may be helpful in devising strategies for developing effective protein formulation and prevention of aggregation of industrially and therapeutically important proteins. Communicated by Ramaswamy H. Sarma

Insights into the Mechanism of Tryptophan Fluorescence Quenching due to Synthetic Crowding Agents: A Combined Experimental and Computational Study.

Intrinsic tryptophan fluorescence spectroscopy is an important tool for examining the effects of molecular crowding and confinement on the structure, dynamics, and function of proteins. Synthetic crowders such as dextran, ficoll, polyethylene glycols, polyvinylpyrrolidone, and their respective monomers are used to mimic crowded intracellular environments. Interactions of these synthetic crowders with tryptophan and the subsequent impact on its fluorescence properties are therefore critically important for understanding the possible interference created by these crowders. In the present study, the effects of polymer and monomer crowders on tryptophan fluorescence were assessed by using experimental and computational approaches. The results of this study demonstrated that both polymer and monomer crowders have an impact on the tryptophan fluorescence intensity; however, the molecular mechanisms of quenching were different. Using Stern-Volmer plots and a temperature variation study, a physical basis for the quenching mechanism of commonly used synthetic crowders was established. The quenching of free tryptophan was found to involve static, dynamic, and sphere-of-action mechanisms. In parallel, computational studies employing Kohn-Sham density functional theory provided a deeper insight into the effects of intermolecular interactions and solvation, resulting in differing quenching modes for these crowders. Taken together, the study offers new physical insights into the quenching mechanisms of some commonly used monomer and polymer synthetic crowders.

Phase Separation and Aggregation of a Globular Folded Protein Small Ubiquitin-like Modifier 1 (SUMO1).

Liquid-liquid phase separation (LLPS) plays a crucial role in cellular organization, primarily driven by intrinsically disordered proteins (IDPs) leading to the formation of biomolecular condensates. A folded protein SUMO that post-translationally modifies cellular proteins has recently emerged as a regulator of LLPS. Given its compact structure and limited flexibility, the precise role of SUMO in condensate formation remains to be investigated. Here, we show the rapid phase separation of SUMO1 into micrometer-sized liquid-like condensates in inert crowders under physiological conditions. Subsequent time-dependent conformational changes and aggregation are probed by label-free methods (tryptophan fluorescence and Raman spectroscopy). Remarkably, experiments on a SUMO1 variant lacking the N-terminal disordered region further corroborate the role of its structured part in phase transitions. Our findings highlight the potential of folded proteins to engage in LLPS and emphasize further investigation into the influence of the SUMO tag on IDPs associated with membrane-less assemblies in cells.

Structural Insights into the Ligand-LsrK Kinase Binding Mode: A Step Forward in the Discovery of Novel Antimicrobial Agents.

LsrK is a bacterial kinase that triggers the quorum sensing, and it represents a druggable target for the identification of new agents for fighting antimicrobial resistance. Herein, we exploited tryptophan fluorescence spectroscopy (TFS) as a suitable technique for the identification of potential LsrK ligands from an in-house library of chemicals comprising synthetic compounds as well as secondary metabolites. Three secondary metabolites (Hib-ester, Hib-carbaldehyde and (R)-ASME) showed effective binding to LsrK, with KD values in the sub-micromolar range. The conformational changes were confirmed via circular dichroism and molecular docking results further validated the findings and displayed the specific mode of interaction. The activity of the identified compounds on the biofilm formation by some Staphylococcus spp. was investigated. Hib-carbaldehyde and (R)-ASME were able to reduce the production of biofilm, with (R)-ASME resulting in the most effective compound with an EC50 of 14 mg/well. The successful application of TFS highlights its usefulness in searching for promising LsrK inhibitor candidates with inhibitor efficacy against biofilm formation.

The impact of lipid packing defects on alpha-synuclein binding to lipid membranes.

Alpha-synuclein (aSyn) is an intrinsically disordered protein found in neurons whose dysfunction is related to the formation of protein aggregates in Parkinson's Disease. While the normal physiological function of aSyn is suspected to be related to the synaptic vesicle cycle, the specific role played by the protein remains unknown. As both the function and dysfunction of aSyn are linked to its interaction with lipid membranes, it is especially important to understand the intricacies of these interactions to discern the differences between normal function and disease progression. While recent work has suggested the importance of lipid packing defects for aSyn-membrane interactions, it is difficult to experimentally establish this dependence on the binding due to the small size and transience of packing defects. In this work, we aim to further understand the effects of lipid packing defects on aSyn-membrane interactions through the use of two complementary binding techniques: tryptophan fluorescence spectroscopy and confocal microscopy. These techniques allow us to access different “binding regimes” for this interaction, which, when fitted with the proper binding equations, can yield distinct parameters that reveal additional binding information beyond the dissociation constant, Kd, for a binding site. Our results highlight the significance of each protein concentration “regime” when studying aSyn-membrane interactions and the defect dependence on the protein binding site density on a lipid membrane. These parameters together provide a more complete picture of aSyn-membrane interactions.

Chitinase is a Potent Insecticidal Molecular Target of Camptothecin and Its Derivatives

Camptothecin (CPT) is a prominent molecule in natural product research because of its application prospects in medicine and agriculture. In this study, CPT and its derivatives were discovered to be competitive inhibitors of group II and group h insect chitinases, both of which are key components of insect chitinolytic systems. CPT and 7-ethyl-10-hydroxycamptothecin (SN-38) inhibited group II chitinase from Ostrinia furnacalis (OfChtII) with Ki values of 5.1 and 2.0 μM, respectively. Results from tryptophan fluorescence spectroscopy, molecular docking analysis, and molecular dynamics simulations revealed that both CPT and SN-38 inhibit OfChtII-C1 by interacting with solvent-exposed tryptophan residues in a substrate-binding cleft. CPT exhibited high insecticidal activity toward the orthopteran pest Locusta migratoria, possibly because of the midgut metabolism of CPT, with only moderate activities toward lepidopteran pests. Even though SN-38 exhibited much lower insecticidal activities than CPT, it still showed higher inhibitory activity toward chitinase. This study reports a new molecular target of CPT and provides insights into molecular design of CPT-based insecticides against different kinds of pests.

Fine-tuning non-covalent interactions between hybrid metal-oxo clusters and proteins.

Interactions between the protein Hen Egg White Lysozyme (HEWL) and three different hybrid Anderson-Evans polyoxometalate clusters - AE-NH2 (δ-[MnMo6O18{(OCH2)3CNH2}2]3-), AE-CH3 (δ-[MnMo6O18{(OCH2)3CCH3}2]3-) and AE-Biot (δ-[MnMo6O18{(OCH2)3CNHCOC9H15N2OS}2]3-) - were studied via tryptophan fluorescence spectroscopy and single crystal X-ray diffraction. Quenching of tryptophan fluorescence was observed in the presence of all three hybrid polyoxometalate clusters (HPOMs), but the extent of quenching and the binding affinity were greatly dependent on the nature of the organic groups attached to the cluster. Control experiments further revealed the synergistic effect of the anionic polyoxometalate core and organic ligands towards enhanced protein interactions. Furthermore, the protein was co-crystallised with each of the three HPOMs, resulting in four different crystal structures, thus allowing for the binding modes of HPOM-protein interactions to be investigated with near-atomic precision. All crystal structures displayed a unique mode of binding of the HPOMs to the protein, with both functionalisation and the pH of the crystallisation conditions influencing the interactions. From the crystal structures, it was determined that HPOM-protein non-covalent complexes formed through a combination of electrostatic attraction between the polyoxometalate cluster and positively charged surface regions of HEWL, and direct and water-mediated hydrogen bonds with both the metal-oxo inorganic core and the functional groups of the ligand, where possible. Hence, functionalisation of metal-oxo clusters shows great potential in tuning their interactions with proteins, which is of interest for several biomedical applications.

Effects of Trimethylamine and Trimethylamine Oxide on Human Serum Albumin Observed by Tryptophan Fluorescence and Absorbance Spectroscopies

Trimethylamine (TMA) is an aliphatic tertiary amine produced by gut microbiota, starting from dietary precursors such as L-choline, L-carnitine and betaine. TMA and its metabolite trimethylamine-N-oxide (TMAO) are elevated in the plasma of cardiovascular disease (CVD) patients. Despite extensive literature on this topic, the scientific community is still divided on which of the two molecules is responsible for the harmful effects on human health. To assess whether the plasma levels of these molecules are also modulated by interactions with macromolecules present in the plasma, the weak bonds between TMA or/and TMAO with human serum albumin (HSA) were studied via molecular docking and spectrofluorimetric assay. The impact of TMA and TMAO on HSA and low-density lipoproteins (LDL) oxidation was also evaluated. Docking analysis shows three main binding sites for TMA and two for TMAO. Spectrofluorimetric results show interactions of HSA with TMA and TMAO; a significant (p = 0.010) decrease in Trp-214 intrinsic fluorescence of HSA was measured starting from the lowest concentrations of both TMA and TMAO (3.26 nM and 29.2 nM, respectively). Furthermore, at all concentrations tested, no significant effect on the formation of carbonyls in HSA was measured (p > 0.05) in the presence of TMA or TMAO. However, 28.6 mM TMAO significantly increased (p < 0.05) the degree of oxidation of LDL, suggesting that TMAO has a pro-oxidant role on LDL.

Magainin 2 and PGLa in bacterial membrane mimics IV: Membrane curvature and partitioning

We previously reported that the synergistically enhanced antimicrobial activity of magainin 2 (MG2a) and PGLa is related to membrane adhesion and fusion. Here, we demonstrate that equimolar mixtures of MG2a and L18W-PGLa induce positive monolayer curvature stress and sense, at the same time, positive mean and Gaussian bilayer curvatures already at low amounts of bound peptide. The combination of both abilities—membrane curvature sensing and inducing—is most likely the base for the synergistically enhanced peptide activity. In addition, our coarse-grained simulations suggest that fusion stalks are promoted by decreasing the free-energy barrier for their formation rather than by stabilizing their shape. We also interrogated peptide partitioning as a function of lipid and peptide concentration using tryptophan fluorescence spectroscopy and peptide-induced leakage of dyes from lipid vesicles. In agreement with a previous report, we find increased membrane partitioning of L18W-PGLa in the presence of MG2a. However, this effect does not prevail to lipid concentrations higher than 1 mM, above which all peptides associate with the lipid bilayers. This implies that synergistic effects of MG2a and L18W-PGLa in previously reported experiments with lipid concentrations >1 mM are due to peptide-induced membrane remodeling and not their specific membrane partitioning.

Adjuvant effect of mesoporous silica SBA-15 on anti-diphtheria and anti-tetanus humoral immune response

Routine immunization against diphtheria and tetanus has drastically reduced the incidence of these diseases worldwide. Anti-diphtheria/tetanus vaccine has in general aluminum salt as adjuvant in its formulation that can produce several adverse effects. There is a growing interest in developing new adjuvants. In this study, we evaluated the efficiency of SBA-15 as an adjuvant in subcutaneous immunization in mice with diphtheria (dANA) and tetanus (tANA) anatoxins as well as with the mixture of them (dtANA). The tANA molecules and their encapsulation in SBA-15 were characterized using Small-Angle X-ray Scattering (SAXS), Dynamical Light Scattering (DLS), Nitrogen Adsorption Isotherm (NAI), Conventional Circular Dichroism (CD)/Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy, and Tryptophan Fluorescence Spectroscopy (FS). The primary and secondary antibody response elicited by subcutaneous immunization of High (HIII) and Low (LIII) antibody responder mice with dANA, tANA, or dtANA encapsulated in the SBA-15 were determined. We demonstrated that SBA-15 increases the immunogenicity of dANA and tANA antigens, especially when administered in combination. We also verified that SBA-15 modulates the antibody response of LIII mice, turning them into high antibody responder. Thus, these results suggest that SBA-15 may be an effective adjuvant for different vaccine formulations.

Spontaneous Refolding of Amyloid Fibrils from One Polymorph to Another Caused by Changes in Environmental Hydrophobicity.

Here, we report a new phenomenon in which lysozyme fibrils formed in a solution of acetic acid spontaneously refold to a different polymorph through a disassembled intermediate upon the removal of acetic acid. The structural changes were revealed and characterized by deep-UV resonance Raman spectroscopy, nonresonance Raman spectroscopy, intrinsic tryptophan fluorescence spectroscopy, and atomic force microscopy. A PPII-like structure with highly solvent-exposed tryptophan residues predominates the intermediate aggregates before refolding to polymorph II fibrils. Furthermore, the disulfide (SS) bonds undergo significant rearrangements upon the removal of acetic acid from the lysozyme fibril environment. The main SS bond conformation changes from gauche-gauche-trans in polymorph I to gauche-gauche-gauche in polymorph II. Changing the hydrophobicity of the fibril environment was concluded to be the decisive factor causing the spontaneous refolding of lysozyme fibrils from one polymorph to another upon the removal of acetic acid. Potential biological implications of the discovered phenomenon are discussed.

Biotinylated peptides substituted with D-amino acids with high stability as anti-anaphylactic agents targeting platelet-activating factor.

Platelet-activating factor (PAF) is an important lipid mediator of anaphylaxis and therefore can be an anti-anaphylactic agent target. Recently, we reported that several synthetic biotinylated peptides containing a Tyr-Lys-Asp-Gly sequence markedly inhibited the bioactivities of PAF in vitro and in vivo; it also inhibited anaphylactic reactions such as hypothermia, hypotension, and vascular permeability in vivo. Here, we report the anti-anaphylactic effects of three biotinylated heptapeptides (peptide 1: H-Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-Asp-OH, peptide 2: H-D -Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-Asp-OH, and peptide 3: H-D -Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-D -Asp-OH). The experiment using tryptophan fluorescence spectroscopy showed that the interaction of peptides 2 and 3 with PAF was larger than that of peptide 1. Experiments using a rat model of hind paw edema showed that peptides 1, 3, and 2 inhibited PAF-induced edema by 67.9%, 69.3%, and 79.3%, respectively. In a mouse model of anaphylaxis, both peptides 2 and 3 showed inhibitory effects on anaphylactic hypothermia, whereas peptide 1 did not. Furthermore, experiments involving in vitro rat plasma stability of peptides showed that both peptides 3 and 2 were more stable in plasma compared to peptide 1 (84.0%, 51.8%, and 0%, remained after 6h, respectively). Our results suggest that both peptides 2 and 3 may show systemic and local inhibitory effects as anti-anaphylactic agents targeting PAF.

Human γS-Crystallin Resists Unfolding Despite Extensive Chemical Modification from Exposure to Ionizing Radiation.

Ionizing radiation has dramatic effects on living organisms, causing damage to proteins, DNA, and other cellular components. γ radiation produces reactive oxygen species (ROS) that damage biological macromolecules. Protein modification due to interactions with hydroxyl radical is one of the most common deleterious effects of radiation. The human eye lens is particularly vulnerable to the effects of ionizing radiation, as it is metabolically inactive and its proteins are not recycled after early development. Therefore, radiation damage accumulates and eventually can lead to cataract formation. Here we explore the impact of γ radiation on a long-lived structural protein. We exposed the human eye lens protein γS-crystallin (HγS) to high doses of γ radiation and investigated the chemical and structural effects. HγS accumulated many post-translational modifications (PTMs), appearing to gain significant oxidative damage. Biochemical assays suggested that cysteines were affected, with the concentration of free thiol reduced with increasing γ radiation exposure. SDS-PAGE analysis showed that irradiated samples form protein-protein cross-links, including nondisulfide covalent bonds. Tandem mass spectrometry on proteolytic digests of irradiated samples revealed that lysine, methionine, tryptophan, leucine, and cysteine were oxidized. Despite these chemical modifications, HγS remained folded past 10.8 kGy of γ irradiation as evidenced by circular dichroism and intrinsic tryptophan fluorescence spectroscopy.

Characterization and functional properties of Maillard reaction products of β-lactoglobulin and polydextrose

The Maillard reaction products (MRPs) between polydextrose (PDX), a popular polysaccharide in formula powder, and β-lactoglobulin (β-LG), a major whey protein, were studied in aggregation degree, structure, hydrophobic, antigenic and antioxidant activity changes of β-LG. Incubation of PDX and β-LG (60 ℃, 79% relative humidity) for up to 72 h yielded MRPs with increases in furosine, UV absorbance, fluorescence intensity and loss of free amino groups. High molecular weight β-LG-PDX MRPs were observed by SDS-PAGE. Circular dichroism spectroscopy revealed negligible change in β-LG secondary structure. Changes in the tertiary structure of β-LG were detected by tryptophan fluorescence spectroscopy consistent with an increase in surface hydrophobicity of heated β-LG-PDX. Antigenicity reduction of β-LG in β-LG-PDX reached its peak when heated for 24 h. After heating for 72 h, DPPH radical-scavenging activity of β-LG-PDX increased by 7.4-fold, and the ferric reducing antioxidant power reached 61.1 µmol ascorbic acid/g protein.

Unraveling the structural basis of urea-induced unfolding of Fasciola gigantica cytosolic malate dehydrogenase

Malate dehydrogenase enzyme plays an essential role in energy metabolism in all eukaryotes. It catalyzes the interconversion of malate to oxaloacetate, producing NAD+/NADH. In the current study, we monitored the urea-induced conformational changes in Fasciola gigantica malate dehydrogenase (FgMDH) structure using multiple spectroscopic techniques combined with all-atom molecular dynamics (MD) simulations. FgMDH showed a two-state cooperative unfolding revealed by intrinsic tryptophan (Trp) fluorescence and CD spectroscopy; however, their non-superimposable curves indicate a partially folded intermediate state formation. The study showed that the unfolding of tertiary structures preceded the secondary structure unfolding. The binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS)at 1.5 M urea confirmed stabilization of the intermediate state. The free energy of stabilization determined from Trp fluorescence and CD spectroscopy were 11.4 and 13.2 kcal.mol−1, respectively. The loss of enzymatic activity was observed before any significant loss in structural components. This suggested that low urea concentrations induce minor structural changes in the substrate or cofactor binding site leading to activity loss. Quenching of fluorescence intensity also provides information on unfolding and activity loss. MD simulations confirmed that FgMDH formed an intermediate state at 1.5 M urea at 300 K, which destabilized at higher urea concentration and temperature due to disrupted hydrogen-bond networks and hydrophobic interactions. We conclude that a partially folded metastable state is stabilized during the unfolding of cytosolic FgMDH. A strong correlation was observed between the data obtained from in-vitro and in-silico approaches.

Tryptophan fluorescence spectroscopy: key tool to study protein denaturation/anti-inflammatory assay

Tryptophan fluorescence spectroscopy: key tool to study protein denaturation/anti-inflammatory assay