Thermodynamics of denaturation of α-chymotrypsinogen A in aqueous urea and alkylurea solutions

The effects of pH, urea, and alkylureas on the thermal stability ofα-chymotrypsinogen A (α-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition ofα-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25°C were calculated. Comparison of these values shows that at 25°C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein molecules. At pH lower than 2.0,α-ctg A still exists in a very stable form, probably the acid-denatured form (A-form).     

The nature of alkylurea and urea denaturation of α-chymotrypsinogen

The optical rotatory dispersion of α-chymotrypsinogen in aqueous solution became less levorotatory upon the addition of ethyl-, propyl-, or butylurea; less negative values for the Moffitt-Yang parameter, α₀, were also obtained. This change in optical rotation of α-chymotrypsinogen induced by the alkylureas was similar in direction and magnitude to that observed for alcohols but was opposite to that caused by unsubstituted urea. It appears, therefore, that the alkylureas share with the alcohols an ability to rearrange α-chymotrypsinogen into a non-native yet regularly ordered conformation. The effectiveness of the alkylureas and alcohols as denaturants for this protein increased in the order ethyl<propyl<butyl derivatives. An identical rank-order was observed for the ability of the alkylureas and alcohols to diminish attractive forces between aliphatic groups, as measured by a model system based upon the extent of aggregation of glass beads coated with methyl groups. These findings indicate that the denaturing action of alkylureas for α-chymotrypsinogen is a function of the substituted aliphatic group and is predominantly hydrophobic in character. Non-hydrophobic interactions of unsubstituted urea with α-chymotrypsinogen appear to be critical for unfolding of the protein to a random-coil configuration.     

Denaturation ofBacillus stearothermophilus α-amylase by urea and detergents

Denaturation ofBacillus stearothermophilus -amylase by urea and detergents was investigated for the purpose of understanding the mechanism of denaturation of this enzyme. The enzyme was extremely resistant to denaturation by detergents at 60° C, either in the presence or absence of added calcium. Addition of EDTA was necessary to obtain denaturation by detergents. The rate of denaturation of the -amylase by urea was strongly dependent on the incubation pH and presence or absence of calcium ions. Calcium-binding groups were shown to have pKa values of 5.5 for exogenous calcium and 4.7 to 4.8 for endogenous calcium. A mechanism is proposed for the denaturation ofBacillus stearothermophilus -amylase.     

Fluorescence studies of the restricted motion of tryptophan in α-cobratoxin

The fluorescence lifetime and rotational correlation time of the single tryptophan residue in -cobratoxin have been measured between pH 2 and 10. The fluorescence decays are non-exponential and give lifetimes that are shorter than normally observed in small proteins (0.3 ns and 1 ns). This emission is consistent with a model in which the tryptophan residue is in slightly different environments in the protein. Fluorescence anisotropy decays show that the tryptophan residue is almost completely immobilised by neighbouring groups in the protein. The range of the wobbling motion is slightly pH dependent and limited to between 5° and 10°.     

Structure and behavior of human α-thrombin upon ligand recognition: thermodynamic and molecular dynamics studies

Thrombin is a serine proteinase that plays a fundamental role in coagulation. In this study, we address the effects of ligand site recognition by alpha-thrombin on conformation and energetics in solution. Active site occupation induces large changes in secondary structure content in thrombin as shown by circular dichroism. Thrombin-D-Phe-Pro-Arg-chloromethyl ketone (PPACK) exhibits enhanced equilibrium and kinetic stability compared to free thrombin, whose difference is rooted in the unfolding step. Small-angle X-ray scattering (SAXS) measurements in solution reveal an overall similarity in the molecular envelope of thrombin and thrombin-PPACK, which differs from the crystal structure of thrombin. Molecular dynamics simulations performed with thrombin lead to different conformations than the one observed in the crystal structure. These data shed light on the diversity of thrombin conformers not previously observed in crystal structures with distinguished catalytic and conformational behaviors, which might have direct implications on novel strategies to design direct thrombin inhibitors.     

Photophysics of diphenyl-pyrazole compounds in solutions and α-synuclein aggregates

Background

Recently diphenyl-pyrazole (DPP) compounds and especially anle138b were found to reduce the aggregation of α-synuclein or Tau protein in vitro as well as in a mouse model of neurodegenerative diseases [1,2]. Direct interaction of the DPPs with the fibrillar structure was identified by fluorescence spectroscopy. Thereby a strong dependence of the fluorescence on the surroundings could be identified [3].

Denaturation of α-lactalbumin and myoglobin by the anionic biosurfactant rhamnolipid

Glycolipid biosurfactants (GBS) are promising environmentally friendly alternatives to chemical surfactants. Surfactants interact with proteins in many applications, often leading to significant changes in protein properties. Given GBS' marked difference in structure compared to traditional chemical surfactants, it is of interest to investigate their impact on protein structure and stability. Here we combine spectroscopic and calorimetric studies to analyze the interactions between the anionic GBS rhamnolipid (RL) and two model proteins α-lactalbumin in the Ca^2 +-free apo-form (αLA) and myoglobin (Mb), whose interactions with traditional surfactants are well known. RL denatures αLA at sub-cmc concentrations (0.1–1 mM) while Mb is only denatured above the cmc, i.e. in the presence of RL micelles. Denaturation leads to increased α-helicity, similar to the effect of SDS. The proteins bind approximately the same amount of RL by weight as SDS. However, RL employs a denaturation mechanism which combines features from non-ionic surfactants (very slow unfolding kinetics and few unfolding steps) with those of SDS (unfolding below the cmc in the case of αLA and the ability to unfold stable proteins in the case of Mb). We ascribe these features to RL's weakly acidic carboxylic head group and complex hydrophobic tail, which lead to a low cmc and low protein affinity. These features restrict the concentration range where RL monomers can bind and denature proteins while still allowing micelles to bind and denature to a significant extent.     

Thirty-four years of dendrochronological studies in Perú: A review of advances and challenges

The development of tree-ring chronologies of tropical trees allows to reconstruct the environmental history of the Neotropics on extensive temporal and spatial scales. This article presents a historic, state-of -the-art overview/review of dendrochronological studies in Perú, a megadiverse country in its flora, types of climate and ecosystems. We reviewed all available information on dendrochronological studies by assessing scientific articles in indexed, and non-indexed journals as well university thesis repositories. Dendrochronological studies began in the late 1980s and have botanically involved 20 families, 34 genera and 52 tree species. The most studied families are Fabaceae (16 studies), Meliaceae (12), Rosaceae (06), and Bignoniaceae (04), and the most studied genera were Cedrela (13), Polylepis (08) and Prosopis (06). The development of chronologies was mainly applied in climatic reconstructions, forest conservation and management. We identify underrepresentation or sampling gaps regarding climatic and geographic complexity. The high tree diversity of Perú constitutes a natural laboratory to develop tree-ring studies to better understand the growth and functioning of tropical tree species, their interaction with climate, and to derive climate reconstructions during the last centuries. This review aims to contribute to the direction of future dendrochronological studies in Perú.     

Secondary structural changes in the intact and the disulfide Bridges cleaved β-lactoglobulin A and B in solutions of urea,guanidine hydrochloride,and sodium dodecyl sulfate

The relative proportions of α-helix, β-sheet, and unordered form in β-lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of β-lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% α-helix and 41% β-sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased β-sheet up to 48% but did not affect the α-helical proportion. The α-helical proportions of nonreduced β-lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the α-helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The β-sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Potential role of α-synuclein in neurodegeneration: studies in a rat animal model

Neuronal protein α-synuclein (α-syn) is an essential player in the development of neurodegenerative diseases called synucleinopathies. A spontaneous autosomal recessive rat model for neurodegeneration was developed in our laboratory. These rats demonstrate progressive increases in α-syn in the brain mesencephalon followed by loss of dopaminergic terminals in the basal ganglia (BG) and motor impairments. The severity of pathology is directly related to the overexpression of α-syn and parallel decrease in dopamine (DA) level in the striatum (ST) of affected rats. The neurodegeneration in this model is characterized by the presence of perikarya and neurites Lewis bodies (LB) and diffuse marked accumulation of perikaryal α-syn in the substantia nigra (SN), brain stem (BS), and striatum (ST) along with neuronal loss. Light and ultrastructural analyses revealed that the process of neuronal degeneration is a 'dying back' type. The disease process is accompanied by gliosis and release of inflammatory cytokines. This neurodegeneration is a multisystemic disease and implicate α-syn as a major factor in the pathogenesis of this inherited autosomal recessive animal model. Decrease dopamine (DA) and overexpression of α-syn in the brain mesencephalon may provide a naturally occurring animal model for Parkinson's disease (PD) and other synucleinopathies that reproduces significant pathological, neurochemical, and behavioral features of the human disease.     

Probing the urea dependence of residual structure in denatured human α-lactalbumin

Backbone ¹⁵N relaxation parameters and ¹⁵N–¹H^N residual dipolar couplings (RDCs) have been measured for a variant of human α-lactalbumin (α-LA) in 4, 6, 8 and 10 M urea. In the α-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala α-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. ¹⁵N R₂ values in some regions of all-Ala α-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental RDC data are compared with RDCs predicted from a statistical coil model and with bulkiness, average area buried upon folding and hydrophobicity profiles in order to identify regions of non-random structure. Residues in the regions corresponding to the B, D and C-terminal 3₁₀ helices in native α-LA show R₂ values and RDC data consistent with some non-random structural propensities even at high urea concentrations. Indeed, for residues 101–106 the residual structure persists in 10 M urea and the RDC data suggest that this might include the formation of a turn-like structure. The data presented here allow a detailed characterization of the non-cooperative unfolding of all-Ala α-LA at higher concentrations of denaturant and complement previous studies which focused on structural features of the molten globule which is populated at lower concentrations of denaturant.     

A differential behavior of α-amylase, in terms of catalytic activity and thermal stability, in response to higher concentration CaCl2

A differential relationship was observed between thermal stability and catalytic activity of α-amylase in the presence of different concentrations of CaCl(2). The enzyme displays optimum catalytic activity in the presence of 1.0-2.0 mM CaCl(2). Further addition of CaCl(2) leads to inhibition of the enzyme, however, at the same time the enzyme gains an additional resistance against thermal denaturation. It was evident that the enzyme is thermodynamically more stable (compared to the active enzyme) in the presence of inhibitory concentration of CaCl(2). For example, the thermal transition temperature (T(m)) of optimally active α-amylase was found to be 64±1°C, whereas, for the less active enzyme (in the presence 10 mM CaCl(2)) the value was determined to be 71±1°C. Similarly, the activation energy of thermal inactivation (Ea) was found to be 228±12 kJ/mol and 291±15 kJ/mol for the optimally active enzyme and the enzyme in the presence of 10 mM CaCl(2), respectively. Biophysical analysis of different states of the enzymes in response to variable calcium level indicates no significant change in the secondary structure in response to different concentration of CaCl(2), however the less active but thermodynamically stable enzyme (in the presence of higher concentration of CaCl(2)) was shown to have relatively more compact structure. The results suggest that the enzyme has separate catalytic and structure stabilizing domains and they significantly vary in their functional attributes in response to calcium level.     

The association−dissociation behavior of the ApoE proteins: kinetic and equilibrium studies

The apolipoprotein E family consists of three major protein isoforms: apolipoprotein E4 (ApoE4), ApoE3, and ApoE2. The isoforms, which contain 299 residues, differ only by single-amino acid changes, but of the three, only ApoE4 is a risk factor for Alzheimer’s disease. At micromolar concentrations, lipid-free ApoE exists predominantly as tetramers. In more dilute solutions, lower-molecular mass species predominate. Using fluorescence correlation spectroscopy (FCS), intermolecular fluorescence resonance energy transfer (FRET), and sedimentation methods, we found that the association?dissociation reaction of ApoE can be modeled with a monomer?dimer?tetramer process. Equilibrium constants have been determined from the sedimentation data, while the individual rate constants for association and dissociation were determined by measurement of the kinetics of dissociation of ApoE and are in agreement with the equilibrium constants. Dissociation kinetics as measured by intermolecular FRET show two phases reflecting the dissociation of tetramer to dimer and of dimer to monomer, with dissociation from tetramer to dimer being more rapid than the dissociation from dimer to monomer. The rate constants differ for the different ApoE isoforms, showing that the association?dissociation process is isoform specific. Strikingly, the association rate constants are almost 2 orders of magnitude slower than expected for a diffusion-controlled process. Dissociation kinetics were also monitored by tryptophan fluorescence in the presence of acrylamide and the data found to be consistent with the monomer?dimer?tetramer model. The approach combining multiple methods establishes the reaction scheme of ApoE self-association.     

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular β-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular β-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.     

The role of β-carotene and vitamin A in atherogenesis: Evidences from preclinical and clinical studies

Atherosclerotic cardiovascular disease (ASCVD) is the principal contributor to myocardial infarction, the leading cause of death worldwide. Epidemiological and mechanistic studies indicate that β-carotene and its vitamin A derivatives stimulate lipid catabolism in several tissues to reduce the incidence of obesity, but their roles within ASCVD are elusive. Herein, we review the mechanisms by which β-carotene and vitamin A modulate ASCVD. First, we summarize the current knowledge linking these nutrients with epidemiological studies and lipoprotein metabolism as one of the initiating factors of ASCVD. Next, we focus on different aspects of vitamin A metabolism in immune cells such as the mechanisms of carotenoid uptake and conversion to the vitamin A metabolite, retinoic acid. Lastly, we review the effects of retinoic acid on immuno-metabolism, differentiation, and function of macrophages and T cells, the two pillars of the innate and adaptive immune response in ASCVD, respectively.This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.     

Fluorescence and molecular dynamics studies of the acetylcholine receptor γM4 transmembrane peptide in reconstituted systems

A combination of fluorescence spectroscopy and molecular dynamics (MD) is applied to assess the conformational dynamics of a peptide making up the outermost ring of the nicotinic acetylcholine receptor (AChR) transmembrane region and the effect of membrane thickness and cholesterol on the hydrophobic matching of this peptide. The fluorescence studies exploit the intrinsic fluorescence of the only tryptophan residue in a synthetic peptide corresponding to the fourth transmembrane domain of the AChR γ subunit (γM4-Trp⁶) reconstituted in lipid bilayers of varying thickness, and combine this information with quenching studies using depth-sensitive phosphatidylcholine spin-labeled probes and acrylamide, polarization of fluorescence, and generalized polarization of Laurdan. A direct correlation was found between bilayer width and the depth of insertion of Trp⁶. We further extend our recent MD study of the conformational dynamics of the AChR channel to focus on the crosstalk between M4 and the lipid-belt region. The isolated γM4 peptide is shown to possess considerable orientational flexibility while maintaining a linear α-helical structure, and to vary its tilt depending on bilayer width and cholesterol (Chol) content. MD studies also show that γM4 also establishes contacts with the other TM peptides on its inner face, stabilizing a shorter TM length that is still highly sensitive to the lipid environment. In the native membrane the topology of the M4 ring is likely to exhibit a similar behavior, dynamically modifying its tilt to match the hydrophobic thickness of the bilayer.     

Molecular level insights into thermally induced α-chymotrypsinogen A amyloid aggregation mechanism and semiflexible protofibril morphology

Understanding nonnative protein aggregation is critical not only to a number of amyloidosis disorders but also for the development of effective and safe biopharmaceuticals. In a series of previous studies [Weiss et al. (2007) Biophys. J. 93, 4392-4403; Andrews et al. (2007) Biochemistry 46, 7558-7571; Andrews et al. (2008) Biochemistry 47, 2397-2403], α-chymotrypsinogen A (aCgn) and bovine granulocyte colony stimulating factor (bG-CSF) have been shown to exhibit the kinetic and morphological features of other nonnative aggregating proteins at low pH and ionic strength. In this study, we investigated the structural mechanism of aCgn aggregation. The resultant aCgn aggregates were found to be soluble and exhibited semiflexible filamentous aggregate morphology under transmission electron microscopy. In addition, the filamentous aggregates were demonstrated to possess amyloid characteristics by both Congo red binding and X-ray diffraction. Peptide level hydrogen exchange (HX) analysis suggested that a buried native β-sheet comprised of three peptide segments (39-46, 51-64, and 106-114) reorganizes into the cross-β amyloid core of aCgn aggregates and that at least ～50% of the sequence adopts a disordered structure in the aggregates. Furthermore, the equimolar, bimodal HX labeling distribution observed for three reported peptides (65-102, 160-180, and 229-245) suggested a heterogeneous assembly of two molecular conformations in aCgn aggregates. This demonstrates that extended β-sheet interactions typical of the amyloid are sufficiently strong that a relatively small fraction of polypeptide sequence can drive formation of filamentous aggregates even under conditions favoring colloidal stability.