Dual behavior of sodium dodecyl sulfate as enhancer or suppressor of insulin aggregation and chaperone-like activity of camel αS1-casein

Sodium dodecyl sulfate (SDS) at low concentrations considerably enhanced insulin aggregation and reduced the chaperone-like activity of purified camel αS₁-casein (αS₁-CN). These observed changes were the result of repulsive electrostatic interactions between both negative charged head groups of SDS and αS₁-CN, and the net negative charge of insulin molecules, resulting in the greater exposure of hydrophobic patches of insulin and its enhanced aggregation. In contrast, enhanced hydrophobic interactions were primarily responsible for the conformational changes observed in insulin and αS₁-CN at high SDS concentrations, resulting in increased binding of SDS and αS₁-CN to insulin and its reduced aggregation.     

Conformational changes of α-lactalbumin induced by the stepwise reduction of its disulfide bridges: The effect of the disulfide bridges on the structural stability of the protein in sodium dodecyl sulfate solution

Four disulfide bridges of bovineα-lactalbumin (α-lact) were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and OSSα-lact, respectively). The original helicity was almost maintained in 3SSα-lact missing only the Cys6-Cysl20 bridge. Upon the reduction of both Cys28-Cys111 and Cys6-Cys120 bridges, various changes occurred in the protein. In particular, the maximum fluorescence of 1-anilinonaphthalene-8-sulfonic acid was observed in this stage. Upon the reduction of all disulfide bridges, the hydrophobic box of the protein, formed by Trp60, Ile95, Tyr103, and Trp104, was disrupted and an internal helical structure was destroyed. The conformation of each derivative was examined mainly in a solution of sodium dodecyl sulfate. In the surfactant solution, the helicity increased from 33% to 37% in 3SSα-lact, from 26% to 31% in 2SSα-lact, and from 18% to 37% in OSSα-lact, as against from 34% to 44% in intactα-lact. On the other hand, the tryptophan fluorescence of each derivative was affected in very low surfactant concentrations, suggesting that the tertiary structure considerably changed prior to the secondary structural change in the surfactant solution.     

Energetics of ligand recognition and self-association of bovine β-lactoglobulin: differences between variants A and B

An understanding of the interplay between structure and energetics is crucial for the optimization of modern protein engineering techniques. In this context, the study of natural isoforms is a subject of major interest, as it provides the scenario for analyzing mutations that have endured during biological evolution. In this study, we performed a comparative analysis of the ligand-recognition and homodimerization energetics of bovine β-lactoglobulin variants A (βlgA) and B (βlgB). These variants differ by only two amino-acid substitutions: 64th (Asp(A) → Gly(B)), which is fully exposed to the solvent, and 118th (Val(A) → Ala(B)), immersed in the hydrophobic core of the protein. Calorimetric measurements revealed significant enthalpic and entropic differences between the isoforms in both binding processes. A structural comparison suggests that a variation in the conformation of the loop C-D, induced by mutation Asp/Gly, could be responsible for the differences in ligand-binding energetics. While recognition of lauric acid was entropically driven, recognition of sodium dodecyl sulfate was both entropically and enthalpically driven, confirming the key role of the ligand polar moiety. Because of a more favorable enthalpy, the dimerization equilibrium constant of βlgB was larger than that of βlgA at room temperature, while the two dimers became similarly stable at 35 °C. The isoforms exchanged the same number of structural water molecules and protons and shared similar stereochemistry at the dimer interface. MD simulations revealed that the subunits of both variants become more flexible upon dimer formation. It is hypothesized that a larger increase of βlgA mobility could account for the dimerization energetic differences observed.     

A study of genetic polymorphisms of milk β-lactoglobulin, αS1-casein, β-casein,and κ-casein in five dairy breeds

Gene frequencies of the milk -lactoglobulin, _S1-casein, -casein, and -casein loci have been estimated from 1663 cows of five dairy breeds. Departure from Hardy-Weinberg equilibrium was found only in the -casein system in Jerseys. However, chance alone could have accounted for this single significant finding. Results of pairwise comparisons among the five breeds of allele frequencies at these milk protein loci indicate that of the 40 possible tests, only six comparisons are not significant at the 5% probability level. It would appear that these breeds are characterizable in terms of the gene frequencies of these milk protein loci. Nonindependent assortment of genotypes among these milk protein loci was also studied. The closely linked casein loci were not independent in almost all the breeds where tests could be carried out. The only exception was between the _S1-casein and -casein loci in Holsteins. -Lactoglobulin was independent of the casein loci in all breeds except Brown Swiss, where it was found to be significantly associated with -casein. Close linkage is proposed as an important factor for maintaining the observed milk protein polymorphisms.This paper represents a portion of a doctoral dissertation submitted by the first author as partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Massachusetts at Amherst.     

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.     

Microenvironment of tryptophan residues in β-lactoglobulin derivative polypeptide-sodium dodecyl sulfate complexes

The changes of microenvironment of tryptophan residues in β-lactoglobulin A and its cyanogen bromide (CNBr) fragments with the binding of sodium dodecyl sulfate (SDS) were studied with measurements of the rates of N-bromosuccinimide (NBS) modification reactions by stopped-flow photometry. Two tryptophan residues of carboxyamidomethylated (RCM) β-lactoglobulin A in the states of their complexes with SDS were clearly distinguishable by their differences in NBS modification rates. We confirmed by experiments with CNBr fragments containing tryptophan residue. The modification rates of Trp 19 in RCM β-lactoglobulin A-SDS complexes were about 10-fold smaller than those expected for tryptophan residues exposed entirely to the aqueous solvent. The Trp 61 was hardly changed. The change of rate constants for Trp 19 was virtually consistent with those observed when N-acetyl-l-tryptophan ethylester was dissolved in SDS micelles. For various species of polypeptide-SDS complexes, all tryptophan residues were reactive to NBS and also, for some of them, the differences in NBS modification rates were observed between tryptophan residues on a common polypeptide chain. These results suggest micellar and heterogeneous bindings of SDS to polypeptides.     

Proteolytic specificity of chicken cathepsin L on bovineβ-casein

The proteolytic specificity of chicken cathepsin L was studied using bovine -casein as substrate. The peptide mixtures obtained after various times of hydrolysis were separated by RP-HPLC and ten peptides were identified. Chicken cathepsin L accepts proline residues in all positions except P ₁ . Looking at the amino acid residues on the amino side of the scissile bond we found three times the Tyr-Pro pair at P ₁ –P ₂ positions and that the S ₁ subsite can interact with modified amino acids such as phosphoserine.Abbreviations RP-HPLC reverse phase high performance liquid chromatography - NMec N-methyl coumarylamide - TEA triethylamine - TFA trifluoroacetic acid     

Implication of C-Terminal Deletion on the Structure and Stability of Bovine β-casein

Bovine β-casein (β-CN) with its C-terminal truncated by chymosin digestion, β-CN-(f1-192), was examined and characterized using circular dichroism (CD) under various temperature conditions. CONTIN/LL analysis of the CD data revealed significant secondary structure disruption in β-CN-(f1-192) relative to its parent protein,β-CN, in the temperature range (5° to 70°C) studied. Near-UV CD spectra indicated significant temperature dependent structural changes. Analytical ultracentrifugation results showed significant reduction but not complete abolishment of self-association in β-CN-(f1-192) compared to whole β-casein at 2°–37°C. Furthermore, binding experiments with the common hydrophobic probe – 8-anilino-1- naphthalene sulfonic acid (ANS) illustrated that β-CN-(f1-192) is nearly incapable of binding to ANS relative to whole β-CN, suggesting a nearly complete open overall tertiary structure brought about by the C-terminal truncation. It has been demonstrated clearly that the tail peptide β-CN-(f193-209) is important in maintaining the hydrophobic core of β-CN but the residual association observed argues for a minor role for other sites as well.     

The effect of context on the folding of β-hairpins

Small β-hairpin peptides have been widely used as models for the folding of β-sheets. But how applicable is the folding of such models to β-structure in larger proteins with conventional hydrophobic cores? Here we present multiple unfolding simulations of three such proteins that contain the WW domain double hairpin β-sheet motif: cold shock protein A (CspA), cold shock protein B (CspB) and glucose permease IIA domain. We compare the behavior of the free motif in solution and in the context of proteins of different size and architecture. Both Csp proteins lost contacts between the double-hairpin motif and the protein core as the first step of unfolding and proceeded to unfold with loss of the third β-strand, similar to the isolated WW domain. The glucose permease IIA domain is a larger protein and the contacts between the motif and the core were not lost as quickly. Instead the unfolding pathway of glucose permease IIA followed a different pathway with β1 pulling away from the sheet first. Interestingly, when the double hairpin motif was excised from the glucose permease IIA domain and simulated in isolation in water it unfolded by the same pathway as the WW domain, indicating that it is tertiary interactions with the protein that alter the motif’s unfolding not a sequence dependent effect on its intrinsic unfolding behavior. With respect to the unfolding of the hairpins, there was no consistent order to the loss of hydrogen bonds between the β-strands in the hairpins in any of the systems. Our results show that while the folding behavior of the isolated WW domain is generally consistent with the double hairpin motif’s behavior in the cold shock proteins, it is not the case for the glucose permease IIA domain. So, one must be cautious in extrapolating findings from model systems to larger more complicated proteins where tertiary interactions can overwhelm intrinsic behavior.     

Role of Asp102 in the enzymatic reaction of bovine β-trypsin: A molecular orbital study

Using the X-ray structure of the complex of bovine β-trypsin with the basic pancreatic trypsin inhibitor, the hydrogen-bond structure consisting of Ser195, His57 and Asp102 is clarified in relation to the mechanism of the enzymatic reaction from an ab initio quantum chemical point of view. Under the influence of the inhibitor, of the three hydrogen bonds involving Ser214, His57 and Ala56 around Asp102, and of the other ionic amino acid residues, Asp102 plays a significant role in lowering the barrier height of the proton transfer from Ser195 to His57 without accepting a proton from His57. The principal cause of the barrier height lowering is the electrostatic interaction.     

Reexamination of the polymeric distributions of κ-casein isolated from bovine milk

-Casein the stabilizing protein of the colloidal milk protein complex was purified from bovine skim milk by the method of McKenzie and Wake (Biochim. Biophys. Acta. 47, 240, 1961). The preparations were examined by sodium dodecyl sulfate gel electrophoresis in the presence and absence of a reducing agent. In the presence of a reducing agent, the -casein migrates as a single low molecular weight band. However, in the absence of a reducing agent, a characteristic pattern of aggregates of varying molecular weight was observed with components ranging from monomer to octamer in integer steps. Densitometry of the Coomassie blue stained gels showed an almost equal distribution of components in each band; carbohydrate staining showed preferential location of sugar residues in lower molecular weight components. Treatment with chymosin (rennin) caused a downward shift in apparent molecular weight for each band with no change in the relative intensity of the Coomassie blue stained bands. Similar gel patterns were observed in whole caseins and partially purified -caseins, indicating that this size distribution is a natural disulfide-linked reporter for the distribution of -casein in casein colloids (micelles).     

The effect of cross-links on the mobility of proteins in dodecyl sulphate–polyacrylamide gels

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate-polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate-polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.     

Action of a cell wall proteinase (PI) from Streptococcus cremoris HP on bovine β-casein

Summary The specificity of a cell wall proteinase (P_I) from Streptococcus cremoris strain HP in its action on bovine -casein was determined. To this end an enzymic digest (pH 6.2; 15° C) of -casein was brought to pH 4.6 and the soluble fraction separated by semi-preparative reversed-phase HPLC. Purified peptides were analyzed by amino acid and end-group analysis. Ten chromatographic components were identified, which together accounted for at least seven cleavage sites all being located in the C-terminal fifty-residue part of -casein. In five cases it concerned a Gln-X or X-Gln peptide linkage. The specificity of this proteinase from S. cremoris HP shows similarity to that reported for a cell wall proteinase from S. lactis NCDO 763 in its action on -casein.Presented at the second FEMS Symposium on Lactic Acid Bacteria held in 1987 at Wageningen, Netherlands     

A molecular dynamics simulation study of the effect of water–graphene interaction on the properties of confined water

The role of water in determining the structure and stability of biomacromolecules has been well studied. In this work, molecular dynamics simulations have been applied to investigate the effect of surface hydrophobicity on the structure and dynamics of water confined between graphene surfaces. In order to evaluate this effect, we apply various attractive/repulsive water–graphene interaction potentials (hydrophobicity). The properties of confined water are studied by applying a purely repulsive interaction potential between water–graphene (modelled as a repulsive r^?12 potential) and repulsive–attractive forces (modelled as an LJ(12-6) potential). Compared to the case of a purely repulsive graphene–water potential, the inclusion of repulsive–attractive forces leads to formation of sharp peaks for density and the number of hydrogen bonds. Also, it was found that repulsive–attractive graphene–water potential caused slower hydrogen bonds dynamics and restricted the diffusion coefficient of water. Consequently, it was found that hydrogen bond breakage and formation rate with the repulsive r^?12 potential model, will increase compared to the corresponding water confined with the LJ(12-6) potential.     

A new assay for ATP sulfurylase based on differential solubility of the sodium salts of adenosine 5′-phosphosulfate and sulfate

A new assay for ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) has been devised, the key feature of which is the quantitative precipitation of the unreacted substrate as Na₂SO₄, in ethanol:water (5:1), leaving the product adenosine 5′-phosphosulfate (APS) in solution with an 80% yield. This separation procedure, coupled with the use of $\text{[}{}^{35}\text{S]SO}{}_4{}^{\mathrm{2?}}$, makes it possible to assay the synthesis of picomole amounts of [³⁵S]APS and thereby determine accurately the initial velocity of the forward reaction catalyzed by ATP sulfurylase. Extracts from tobacco cells synthesized [³⁵S]APS and a negligible amount of one unknown sulfur compond, but they did not synthesize 3′-phosphoadenosine 5′-[³⁵S]phosphosulfate (PAPS) under the assay conditions adopted, so that the ³⁵S remaining in solution after ethanol precipitation of $\text{[}{}^{35}\text{S]SO}{}_4{}^{\mathrm{2?}}$ is a valid assay of the ATP sulfurylase reaction in these extracts. This assay is simple, rapid, and suitable for assaying multiple samples. Extracts of four additional species of plants were incubated under the same conditions, and each catalyzed the synthesis of [³⁵S]APS according to this assay. The assay is suitable for use with the enzyme from any organism, provided that the product APS is not acted upon by another enzyme.     

Studies of the effect of organic solvents on the stability of β-glucosidase fromFusarium oxysporum

Summary The effect of organic solvents on the stability of -glucosidase in a powder form, isolated fromFusarium oxysporum, has been studied using several organic solvents of different degree of hydrophobicity. It was found that -glucosidase remains quite stable after a prolonged incubation in the presence of most of the organic solvents used, even at temperatures as high as 70°C. Only dimethyformamide (DMF) and tetrahydrofuran (THF) reduce considerably the enzyme activity in a short preincubation period. Studies on the effect of the pH of the buffer used prior to lyophilization, as well as of exogenous added water to the incubation mixture, on enzyme stability show that it is more stable in pH 5.0 and in the lowest water content. In addition it was found that the presence of glucose in the lyophilization procedure gives a significant protection to the enzyme when it is incubated for 30 h in pentanol and n-hexane.     

The Influence of Heparan Sulfate on Breast Amyloidosis and the Toxicity of the Pre-fibrils Formed by β-casein

The Protein Journal - Heparan sulfate (HS) as a mediator is usually involved in both inflammation and fibrosis. Besides, pre-fibrils are the intermediates of amyloid fibrils that usually cause cell...