Effect of temperature and pH on the β–helix transition of poly(L-lysine) in sodium dodecyl sulfate solution

The conformational phase diagram of poly(L -lysine) (4.6 × 10^?4 M, residue) in sodium dodecyl sulfate (1.6 × 10^?2 M) solution was constructed from circular dichroism results at various temperatures and pH's. Poly(L -lysine)–sodium dodecyl sulfate complexes undergo a β–helix transition upon raising the pH of the solution. The transition pH tends to shift downward at elevated temperatures. No helix–β transition can be detected for poly(L -lysine) in sodium dodecyl sulfate solution (pH > 11) even after 1-hr heating at 70°C. This is in marked contrast with uncharged poly(L -lysine) solution without sodium dodecyl sulfate, which is converted into the β-form upon mild heating of the solution above 50°C.     

Estimation of the circular dichroism exhibited by statistical coils of poly(L-alanine) and unionized poly(L-lysine) in water

The circular dichroism of Ac-(Ala)_x-OMe and H-Lys-(Lys)_x-OH with x = 1, 2, 3, and 4 has been measured in aqueous solutions. The oligomers with x = 4 show similar circular dichroism spectra in water when the lysyl amino groups are protonated, and they respond in similar fashion to heating and to sodium perchlorate. Both oligomers at 15°C exhibit a positive circular dichroism band at 217–218 nm, which is eliminated by the isothermal addition of 4 M sodium perchlorate or by heating. The positive circular dichroism of the lysine oligomer is also eliminated when the pH is elevated to deprotonate the amino groups. Positive circular dichroism is still observed for Ac-(Ala)₄-OMe at elevated pH. Circular dichroism spectra have been estimated for poly(L -alanine) and poly(L -lysine) as statistical coils under the above conditions, based on the trends established with the oligomers. Poly(L -lysine) and poly(L -alanine) are predicted to exhibit similar circular dichroism behavior in aqueous solution so long as the lysyl amino groups are protonated. The circular dichroism of the statistical coil of poly(L -lysine), but not poly(L -alanine), is predicted to change when the pH is elevated sufficiently to deprotonate the lysyl amino groups. These results suggest that the unionized lysyl side chains participate in interactions that are not available to poly(L -alanine). Hydrophobic interactions may occur between the unionized lysyl side chains. Protonation of the lysyl amino groups is proposed to disrupt these interactions, causing poly(L -alanine) and protonated poly(L -lysine) to have similar circular dichroism properties.     

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

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

Optical effects of sodium dodecyl sulfate treatment of the isolated light harvesting complex of higher plants

The light-harvesting complex (LHC) of higher plants isolated using Triton X-100 has been studied during its transformation into a monomeric form known as CPII. The change was accomplished by gradually increasing the concentration of the detergent, sodium dodecyl sulfate (SDS). Changes in the red spectral region of the absorption, circular dichroism (CD), and linear dichroism spectra occurring during this treatment have been observed at room temperature. According to a current hypothesis the main features of the visible region absorption and CD spectra of CPII can be explained reasonably successfully in terms of an exciton coupling among its chlorophyll (Chl) b molecules. We suggest that the spectral differences between the isolated LHC and the CPII may be understood basically in terms of an exciton coupling between the Chl b core of a given CPII unit and at least one of the Chla's of either the same or the adjacent CPII. We propose that this Chl a-Chl b coupling existing in LHC disappears upon segregation into CPII, probably as a result of a detergent-related overall rotation of the strongly coupled Chl b core which changes the relative orientations of the two types of pigments and thus the nature of their coupling.Abbreviations Chl Chlorophyll - CD Circular dichroism - LD Linear dichroism - LHC Light-harvesting complex - SDS Sodium dodecyl sulfate - CPII A solubilized form of LHC obtained with SDS polyacrylamide gel electrophoresis Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Presence of an amphipathic helical segment and its relationship to biological potency of calcitonin analogs

The conformational properties of a number of calcitonin analogs were studied by circular dichroism. The ability of dimyristoylphosphatidylglycerol, lysophosphatidylcholine or sodium dodecyl sulfate to induce the formation of more highly ordered structures in these peptides was also assessed by circular dichroism. In all cases sodium dodecyl sulfate induced the largest change in the circular dichroism spectra of the peptides. Salmon calcitonin and its analogs were slightly more helical in the presence of the anionic phospholipid than in the presence of the zwitterionic detergent lysophosphatidylcholine while the reverse is true for human calcitonin and its analogs. Some of the calcitonin analogs convert turbid suspensions of phosphatidylglycerol to a clear solution from which the phospholipid is no longer readily sedimentable by centrifugation. Several of the physical properties of these peptides could be correlated with their biological activity. Generally peptides which showed no hypocalcemic activity had the least negative mean residue ellipticities at 222 nm. Only biologically active analogs were able quantitatively to solubilize dimyristoyl-phosphatidylglycerol and in this solubilized form the peptides have a higher helical content. More active derivatives exhibit larger increases in helix content in the presence of this phospholipid. Inactive analogs had the least negative mean residue ellipticities at 222 nm in the presence of lysophosphatidylcholine or sodium dodecyl sulfate. Thus, the ability of a calcitonin analog to form structures of higher helical content in the presence of amphiphiles is a requirement for the analog to exhibit high potency in assays of biological activity.     

Conformational changes induced in ionized poly(L-arginine) and poly(L-histidine) by sodium dodecyl sulfate

Circular dichroism spectra have been obtained for cationic poly(L -arginine) and poly(L -histidine) in aqueous solutions containing varying amounts of sodium dodecyl sulfate. The detergent induces a disorder-order transition in both polypeptides. In each case the transition is cooperative and occurs when the ratio of detergent to amino acid residue is near unity. The ordered structure formed by poly(L -arginine) is readily identifiable as an α helix. Poly(L -histidine) appears to form a β structure in which the 211-nm electronic absorption band of the imidazole group exhibits significant rotatory strength.     

Circular dichroism of a membrane protein of Neurospora crassa

Membrane protein of a mutant of Neurospora crassa has been isolated in aqueous sodium phosphate buffer without employing a chemical agent such as urea, detergent, or organic solvent. The protein moved in a single band on sodium dodecyl sulfate gel electrophoresis and as well on non-SDS gel electrophoresis. The circular dichroism spectrum of the protein exhibited the characteristic low ellipticity but no red shift.     

Two-step folding of recombinant mitochondrial porin in detergent

Precise information regarding the transmembrane topology of mitochondrial porin is essential for understanding the mechanisms by which this protein functions. Porin acts as a channel in the outer membrane and interacts with small solutes and proteins to regulate mitochondrial function. The acquisition of high-resolution structural data requires a method of maintaining high concentrations of unaggregated, properly folded porin. In the current studies, several mixed detergent systems were analyzed for their ability to fold Neurospora mitochondrial porin expressed in and isolated from Escherichia coli. A mixture of sodium dodecyl sulfate and dodecyl-β-D-maltopyranoside in a 1:6 molar ratio supports a β-strand-rich conformation. In this state, the two tryptophan residues in the protein reside in hydrophobic environments, and about half of the nine tyrosines are solvent exposed. Most importantly, heat-labile tertiary contacts, as detected by near-UV circular dichroism spectropolarimetry, in the sodium dodecyl sulfate/dodecyl-β-D-maltopyranoside-solubilized porin are very similar to those of the protein following functional reconstitution into liposomes. Similarly, both forms are protease resistant. Thus, a method has been identified with the potential to solubilize high concentrations of mitochondrial porin in a state virtually indistinguishable from the membrane-embedded form.     

Photoresponsive polymers: Azobenzene-containing poly(L-lysine)

Poly(L -lysine) was reacted with various azo-reagents, including p-phenylazobenzoic acid, p-phenylazobenzoyl chloride, and p-phenylazobenzoic N-hydroxy-succinimide ester, to give polypeptides containing 5–44 mol % azobenzene units in the side chains. The conformation of the azo-modified polypeptides was investigated in connection with their photochromic behavior caused by the trans ? cis photoisomerization of the azo groups present in the side chains. In methanol/water solvent mixture, the 20% azo-poly(L -lysine) adopts the α-helix conformation. The helix stability was found to be higher when the azo side chains are in cis than when they are in trans configuration. So irradiation at 340 nm (trans-to-cis isomerization), and alternately at 450 nm (cis-to-trans isomerization), produced reversible variations of the α-helix content. In hexafluoro-2-propanol/water/sodium dodecyl sulfate mixture, the 43% azo-poly(L -lysine) adopts a β-structure, as indicated by CD spectra. Irradiation at 340 nm caused the disruption of the β-structure and promoted the α-helix conformation. The effect was reversed upon irradiation at 450 nm. The photoinduced β ? helix change was explained on the basis of the different geometry and hydrophobic character of the trans and the cis azobenzene units.     

The thermal induced helix--beta transition of poly(N epsilon-methyl-L-lysine) and poly(N delta-ethyl-L-ornithine) in aqueous solution

Uncharged poly(N^ε-methyl-L -lysine) (PMLL) and its isomer, poly(N^δ-ethyl-L -ornithine) (PELO), in alkaline solution (pH ca. 12) undergo a helix-to-β transition upon mild heating at 50°C or higher in a manner similar to that of poly(L -lysine) (PLL). The rate of conversion follows the order: PMLL < PELO < PLL. The helix can be regenerated upon cooling near zero degrees, for instance, after more than 12 hr at 2°C. At concentrations less than 0.02% the β form is intramolecular, but at higher concentrations both intra- and intermolecular β forms are generated. Poly(N^δ-methyl-L -ornithine) (PMLO), an isomer of PLL, behaves like poly(L -ornithine); uncharged PMLO in alkaline solution is partially helical and becomes disordered at elevated temperatures.     

Effect of ethyleneoxide groups of anionic surfactants on lipase activity

The use of enzymes in laundry and dish detergent products is growing. Such tendency implies dedicated studies to understand surfactant‐enzyme interactions. The interactions between surfactants and enzymes and their impact on the catalytic efficiency represent a central problem and were here evaluated using circular dichroism, dynamic light scattering, and enzyme activity determinations. This work focuses on this key issue by evaluating the role of the ethyleneoxide (EO) groups of anionic surfactants on the structure and activity of a commercial lipase, and by focusing on the protein/surfactant interactions at a molecular level. The conformational changes and enzymatic activity of the protein were evaluated in the presence of sodium dodecyl sulfate (SDS also denoted as SLE₀S) and of sodium lauryl ether sulfate with two EO units (SLE₂S). The results strongly suggest that the presence of EO units in the surfactant polar headgroup determines the stability and the activity of the enzyme. While SDS promotes enzyme denaturation and consequent loss of activity, SLE₂S preserves the enzyme structure and activity. The data further highlights that the electrostatic interactions among the protein groups are changed by the presence of the adsorbed anionic surfactants being such absorption mainly driven by hydrophobic interactions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1276–1282, 2016     

Spatial structure of secretin molecule

By conformational analysis and circular dichroism the structure of peptide hormone secretin and its shortened N-terminal fragments in different solvents (water, aqueous solutions of alpha-L-phosphatidic acid and sodium dodecyl sulfate) have been studied. The results obtained by the two methods are compared.     

The random coil leads to beta transition of copolymers of L-lysine and L-valine: potentiometric titration and circular dichroism studies.

A series of copolymers of L -lysine and L -valine [poly(L -lysine^f L -valine^100-f)] containing 0–13% L -valine have been studied, in 0.10M KF solution, using potentiometric titration and circular dichroism spectroscopy. Incorporation of increasing amounts of valine into the copolymers favors β-sheet formation over α-helix formation at high pH and room temperature. The titrations were analyzed using the method of Zimm and Rice and the partial free energy (ΔG⁰_cβ) for the coil-to-β-sheet transition for valine is estimated at 900 cal/mole at 25°C. From the temperature dependence of the free energy, the partial enthalpy, ΔH⁰_cβ, and entropy, ΔS⁰_cβ, of the transition for valine is estimated to be 854 cal/mole and 6.0 e.u., respectively. The corresponding partial thermodynamic parameters for L -lysine are in agreement with published results. The fraction of β-sheet versus pH has been calculated for poly(L -lysine^86.8 L -valine^13.2) at 25.0°C using the titration data; data obtained from circular dichroism spectroscopy for the same copolymer are in good accord. It is concluded from these results that L -valine is a very strong β-sheet forming amino acid. Furthermore, these results indicate that the Zimm–Rice method is applicable to transitions between the coil and β-sheet states for a polypeptide containing two different residues.     

Towards crystallization of hydrophobic myelin glycoproteins: P0 and PASII/PMP22

The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peripheral nervous system of vertebrates is the example of a membrane for which neutral or "gentle" detergents are not even strong enough to solubilize its proteins. In contrast, sodium- or lithium-dodecyl sulfate is very effective. We solubilized myelin membrane in 2%(w/v) sodium dodecyl sulfate, followed by chromatographic purification of the hydrophobic myelin glycoproteins P0 and PASII/PMP22, and finally, we have exchanged the sodium dodecyl sulfate bound to protein for other neutral detergents using ceramic hydroxyapatite column. Theoretically, we should easily exchange sodium dodecyl sulfate for any neutral detergent, but for some of them, the solubility of myelin glycoproteins is low. To monitor the potential variability in the secondary structure of glycoproteins, we have used circular dichroism. Sodium dodecyl sulfate seems to be the appropriate detergent for the purpose of purification of very hydrophobic glycoproteins, since it can be easily exchanged for another neutral detergent.     

The hydrophobic nature of the pig intrinsic factor receptor in the intestine

The pig intestinal intrinsic factor receptor has been isolated and dissociated into its α and β subunits. The β subunit was found to be more hydrophobic than the α subunit. In a detergent solution only the α subunit was accessible to digestion with papain. The whole isolated receptor was introduced into artificial single bilayer liposomes where is apparently was randomly oriented. Liposomes containing the receptor were digested with papain and the polypeptide segments that stayed in the lipid fraction were extracted and analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Four species were found with $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ values of 23 000, 45 000, 70 000 and 86 000.     

Characterization of the apolipoprotein B polypeptide of human plasma low density lipoprotein in detergent and denaturation solutions.

Apolipoprotein B, the polypeptide moiety of human serum low density lipoprotein, is subject to degradation (as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) both in the intact particle and after delipidation. Protease inhibitors, sodium azide, and nitrogen saturation did not influence the rate or degree of degradation. Lipid-free apolipoprotein B prepared by gel exclusion chromatography in sodium dodecyl sulfate bound a limited number of detergent molecules (up to 300) in monomeric sodium dodecyl sulfate solutions; circular dichroic spectra of this complex were similar to spectra of the intact lipoprotein. Near the critical micelle concentrations, a large, cooperative increase in detergent binding occurred, accompanied by circular dichroic changes indicating increased alpha helicity. By sucrose density centrifugation, lysopalmitoyl phosphatidylcholine could be substituted for the anionic detergent; about 300 mol of lysolipid were bound to the polypeptide. Replacement of detergent with guanidine hydrochloride by dialysis produced a soluble polypeptide with no ordered structure at denaturant concentrations above 7 M. At lower guanidine hydrochloride concentrations, structural elements were regained in a broad, reversible transition. It appears that apolipoprotein B is an easily degraded polypeptide with regions resembling water-soluble proteins but other regions which interact with lipid (or synthetic amphiphiles) and produce an overall insolubility in aqueous solution in the absence of amphiphilic ligands.     

Selective,reversible inhibition of the lactose phosphotransferase system of Staphylococcus aureus by dodecyl sulfate and deoxycholate

Low concentrations of sodium dodecyl sulfate (0.015%) and sodium deoxycholate (0.33%) completely inhibit phosphorylation of β-galactosides by the lactose phosphotransferase system of Staphylococcus aureus. Inhibition is reversible, even after prolonged detergent treatment. Phosphorylation of methyl-α-glucoside by the same preparations is only slightly inhibited by 0.015% dodecyl sulfate. The membrane-bound component, Enzyme IF^lac, is not solubilized by 0.015% dodecyl sulfate, nor is its ability to bind [¹⁴C]lactose affected. The results are consistent with hypotheses of selective binding of anionic detergent to Enzyme II^lac or to Factor III^lac, the detergent serving in the latter case as a membrane analog.     

Oligomerization of a peptide derived from the transmembrane region of the sodium pump gamma subunit: effect of the pathological mutation G41R

The Na,K-ATPase, or sodium pump, is a ubiquitously expressed membrane-bound enzyme that controls the transmembrane (TM) gradients of sodium and potassium ions in animal cells. The enzyme comprises two subunits, alpha and beta, and in the kidney, is also associated with a small single-spanning membrane protein, the gamma subunit. This 65 amino acid residues protein has been linked to a form of dominant renal hypomagnesaemia resulting from substitution of a highly conserved glycine residue (Gly41) to arginine residue. In order to characterize the quaternary structure of the gamma subunit, and effects of the G41R mutation thereupon, we synthesized a series of peptides (wild-type and mutant) that span the gamma subunit TM region. Using circular dichroism spectroscopy, we show that the 32-amino acid residue peptides are random coils in aqueous buffer but spontaneously adopt an alpha-helical conformation in the presence of detergent micelles (sodium dodecyl sulfate, SDS, and perfluorooctanoate, PFO). Furthermore, fluorescence resonance energy transfer experiments, combined with polyacrylamide gel electrophoresis, demonstrate that while gamma-TM does not self-associate in SDS, it forms oligomers in PFO, a detergent that tolerates relatively weak associations between membrane proteins. Importantly, oligomerization of gamma-TM is abrogated in a peptide that contains either the disease-causing mutation G41R, or the more conservative mutation G41L. On the other hand, a peptide that contains a Gly-to-Arg substitution on a different face of the helix, at position 35, retains its ability to oligomerize. Our results provide evidence for a link between renal hypomagnesaemia and gamma subunit oligomerization.     

Size-exclusion chromatographic study of the reduction of recombinant hepatitis B surface antigen

The reduction of the P. pastoris-derived hepatitis B surface antigen (HBsAg) has been investigated by size exclusion chromatography performed in a detergent solution containing 0.3% sodium dodecyl sulfate (SDS) and 0.1 M Tris–HCl, pH 7.0. The HBsAg, reduced under different conditions and passed through the TSK G4000 SW column (600×7.5 mm I.D.) at 0.9 ml min⁻¹, was resolved into two peaks corresponding to the reduced, monomeric, and non-reduced forms, respectively. Under these conditions, the antigen fraction corresponding to the HBsAg dimer can be separated and completely reduced to monomers by repeated reductive treatment with simultaneous lipid removal. The efficiency of reduction was maximal after sample treatment with an equal volume of a solution containing 417 mM dithiothreitol, 4.2% (w/v) SDS and 16% (v/v) 2-mercaptoethanol. In conclusion, complete reduction of recombinant HBsAg to monomer subunits is possible and depends on the efficiency of lipid removal during the reductive treatment.     

Electrophoretic behavior of protein dodecyl sulfate complexes in the presence of various amounts of sodium dodecyl sulfate.

This report describes the relationship between the amount of sodium dodecyl sulfate present in a sample solution and the electrophoretic mobility of the protein-dodecyl sulfate complexes. In order to determine the extent of any conformational changes in the proteins and to establish a correlation between any of these structural changes and the electrophoretic behavior, visible absorption spectra and circular dichroism spectra were obtained for heme proteins in the presence of the same amounts of surfactants as used in electrophoresis.From the results obtained, it is apparent that the amount of sodium dodecyl sulfate present in the sample solution must be taken into consideration when performing a separation. Optimum experimental conditions are chosen for attaining enhanced separation and a maximized linear range of molecular weights of proteins that can be accurately determined.