Multiphasic kinetics of myoglobin/sodium dodecyl sulfate complex formation

We have carried out a kinetic analysis of the conformational changes that myoglobin (Mb) undergoes in the presence of the anionic surfactant sodium dodecyl sulfate (SDS). The time-resolved results have been combined with steady-state circular dichroism (CD) and resonance Raman (RR) spectroscopy. Time-resolved absorption spectra indicate that SDS induces changes in the heme coordination with the formation of three different Mb species, depending on SDS concentration. The formation of the Mb/SDS complex involves three or four phases, depending on surfactant concentration. The kinetic data are analyzed assuming two modes of interaction according to whether SDS is monomeric or micellar. The two pathways are separated but interconnected through free Mb. At the lowest concentrations a six-coordinated, low-spin form dominates. Two distinct five-coordinated species are formed at higher SDS concentrations: one is a protein-free heme and the other reequilibrates slowly with the six-coordinated, low-spin form. The resulting complexes have been characterized by CD and RR. In addition, CD spectra show that the local changes in the heme environment are coupled to changes in the protein structure.     

Stop-flow kinetics studies of the interaction of surfactant, sodium dodecyl sulfate, with acid-denatured cytochrome c

Interactions of sodium dodecyl sulfate (SDS) at submicellar and micellar concentration, with the globular protein, horse heart cytochrome c, at low pH have been shown to stabilize two molten globule-like intermediates. These dynamic studies were performed using far-UV, near-UV, and Soret-band circular dichroism (CD) as well as fluorescence methods. Stopped-flow CD and fluorescence studies of acid-denatured cytochrome c refolding with SDS were performed at both submicellar and micellar concentrations. Distinctive refolding mechanisms (from analysis of both CD and fluorescence) were found under these two conditions, and an obvious refolding intermediate was evident in the fluorescence traces. In addition, stopped-flow CD in the Soret region showed multistep kinetics, suggesting that the spectral changes in this region are not only solvent effect related but also connected with the change of secondary structure. A possible folding mechanism is proposed to rationalize the kinetics results.     

Inactivation and conformational changes of lactate dehydrogenase from porcine heart in sodium dodecyl sulfate solutions

The inactivation and conformational changes of porcine heart lactate dehydrogenase (LDH) have been studied in sodium dodecyl sulfate (SDS) solutions. Increasing SDS concentration led to a quick and concentration-dependent inhibition of the enzyme, with complete inactivation within 5 min in the presence of 1.0 mM SDS. Meanwhile, fluorescence emission and circular dichroism spectra were used to follow the conformational changes of the enzyme during this process, concurrently showing that SDS less than 1.0 mM induced only limited conformational changes to LDH. The above results are in accordance with the suggestion by Tsou (Trends Biochem. Sci. 11 (1986) 427; Science 262 (1993) 380) that the active site usually be more flexible than the enzyme molecule as a whole. Furthermore, the results of polyacrylamide gel electrophoresis (PAGE) implied that unfolding intermediates were presented in the above process. When the SDS concentration used to treat LDH was increased, the bands of native enzyme on native PAGE faded and finally almost disappeared. Meanwhile, multiple bands with lower mobility but no activity emerged behind and enhanced correspondingly. Fast protein liquid chromatography indicated that dissociation occurred during the course of denaturation. The reasons for the above phenomena have been discussed. It was suggested that SDS, binding to LDH to form different LDH-SDS complexes, conferred an array of different unfolding states over the enzyme, and in turn resulted in the formation of the multiple bands on the native PAGE.     

HPLC and tandem detection to monitor conformational properties of biopharmaceuticals

High-performance liquid chromatography (HPLC) with UV, circular dichroism (CD) and intrinsic fluorescence detection was applied to monitor conformational properties of recombinant human interferon alpha2b when performing size exclusion chromatography (SEC) and reversed-phase HPLC (RP-HPLC). In this way native conditions during SEC and structural changes of the protein during RP-HPLC were demonstrated. These results were confirmed by stand-alone fluorescence and CD measurements. With respect to HPLC tandem detection, the fluorescence detector compared favourably to the UV and CD detector regarding linearity, sensitivity and selectivity. SEC combined with intrinsic fluorescence scanning detection permits conformational analysis of small amounts of aggregates in the presence of excess native monomeric protein. In conclusion, HPLC with on-line UV and intrinsic fluorescence detection provides a promising concept for analysing the amount and conformational properties of a biopharmaceutical and its impurities.     

Studies on pig serum lipoproteins. V. Optical properties of low density lipoproteins.

The circular dichroism (CD), optical rotatory dispersion (ORD), and fluorescence emission spectra of two subfractions of pig serum low density lipoproteins (LDL1 and LDL2) were compared. The contribution of the carbohydrate moiety to the CD and ORD spectra was estimated on the basis of data obtained from isolated glycopeptides and the constituent monosaccharides. The carbohydrate moiety had no effect on the conformation of the protein moieties of LDL1 and LDL2 (apoLDL1 and apoLDL2). However, the intensities of the observed extrema in the CD and ORD spectra of the glycopeptides were greater than those expected from the monosaccharide composition. This suggests the existence of secondary structure in the carbohydrate moiety. In contrast to the carbohydrate moiety, the contribution of the lipid moiety to the CD and ORD spectra could not be neglected. When the effect of the lipid moiety was subtrated from the CD and ORD spectra, the spectra due to apoLDL1 and apoLDL2 were quite similar. Delipidation in the presence of sodium dodecyl sulfate (SDS) induced an increase in the content of disordered structure and alpha-helix accompanied by a decrease in the beta-structure. In the presence of SDS, marked quenching occurred in the fluorescence emission spectra with a blue shift of the maximum emission wavelength from 332 to 326 nm. ApoLDL1 and apoLDL2 showed quite similar SDS-induced conformational transitions. The secondary structures of apoLDL1 and apoLDL2 in the native lipoproteins were stable to changes of pH and temperature. However, this stability was lost in the presence of SDS. These results suggest the importance of the lipid moiety in maintaining the native secondary structures of LDL1 and LDL2. From the overall similarity of the optical properties of apoLDL1 and apoLDL2, we conclude that the secondary structures of apoLDL1 and apoLDL2 are identical.     

Spectroscopic characterization of the EF-hand domain of phospholipase C delta1: identification of a lipid interacting domain

The interaction of the isolated EF-hand domain of phospholipase C delta1 with arachidonic acid (AA) was characterized using circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectral changes indicate that AA binds to the EF domain. The near-UV CD spectra suggest that the orientations of aromatic residues in the peptide are affected when AA binds to the protein. The fluorescence of the single intrinsic tryptophan located in EF1 was enhanced by the addition of dodecylmaltoside (DDM) and AA suggesting that this region of the protein is involved in hydrophobic interactions. In the presence of a low concentration of DDM it was found that AA induced a change in fluorescence resonance energy transfer, which is indicative of a conformational change. The lipid induced conformational change may play a role in calcium binding because the isolated EF-hand domain did not bind Ca2+ in the absence of lipids, but Ca2+-dependent changes in the intrinsic tryptophan emission were observed when free fatty acids were present. These studies identify specific EF-hand domains as allosteric regulatory domains that require hydrophobic ligands such as lipids.     

Conformational changes in histone IV

Conformational change of histone IV, induced by phosphate, have been investigated by observing the intrinsic fluorescence of tyrosine residues and circular dichroism (CD). There is a fast conformational change upon the addition of phosphate, followed by a slow process with time constants in the range of minutes to hours depending upon both the phosphate and histone concentrations. The CD results indicate α-helix formation in the fast process, and β-sheet formation in the slow one, although other secondary and tertiary structural changes also may occur. The histone concentration dependence of the fast process is consistent with dimerization. Divalent phosphate is about ten times more effective than monovalent phosphate in inducing conformational changes. All of the changes are reversible.     

Spectroscopic analysis of the intrinsic chromophores within small multidrug resistance protein SugE

Small multidrug resistance (SMR) protein family member, SugE, is an integral inner membrane protein that confers host resistance to antiseptic quaternary cation compounds (QCC). SugE studies generally focus on its resistance to limited substrates in comparison to SMR protein EmrE. This study examines the conformational characteristics of SugE protein in two detergents, sodium dodecyl sulphate (SDS) and dodecyl maltoside (DDM), commonly used to study SMR proteins. The influence of cetylpyridinium (CTP) and cetrimide (CET) using SugE aromatic residues (4W, 2Y, 1F) as intrinsic spectroscopic probes was also determined. Organically extracted detergent solubilized Escherichia coli SugE protein was examined by SDS-Tricine PAGE and various spectroscopic techniques. SDS-Tricine PAGE analysis of SugE in either detergent demonstrates the protein predominates as a monomer but also dimerizes in SDS. Far-UV region circular dichroism (CD) analysis determined that the overall α-helix content SugE in SDS and DDM was almost identical and unaltered by QCC. Near-UV region CD, fluorescence, and second-derivative ultraviolet absorption (SDUV) indicated that only DDM-SugE promoted hydrophobic environments for its Trp and Tyr residues that were perturbed by QCC addition. This study identified that only the tertiary structure of SugE protein in DDM is altered by QCC.     

Alkalin titrations of human somatotropin, human choriomammotropin and ovine prolactin by circular dichroism and fluorescence.

Structural transitions occurring during the alkalin titration of human somatotropin, human choriomammotropin, and ovine prolactin have been investigated by means of circular dichroism and fluorescence emission spectra. Human somatotropin exhibited an isodichroic point at 287 nm, with all spectral changes being reversed upon back titration from pH 12.50 to pH 8.0. Fluorescence quenching as a function of pH produced a simple sigmoidal curve. Human choriomammotropin exhibited an isodichroic point at 288 nm. The fluorescence and circular dichroism spectra of this protein were found to be reversible between pH 8.0 and 11.0. However, on titration above pH 11, the isodichroic point and the reversibility of the circular dichroism spectra were lost. This conformational transition was accompanied by a sharp increase in fluorescence quantum yield. The circular dichroism spectra of ovine prolactin showed essentially no change on titration to pH 11.0. However, between pH 11.0 and 12.0, a sharp conformational transition was observed similar to that seen in human choriomammotropin, but not exhibiting the same increase in fluorescence quantum yield. The fluorescence titration of prolactin was found to be essentially reversible upon back titration from pH 12.5, although the circular dichroism spectra were not reversible from this pH.     

Kinetic Studies on the Interaction of Ferricytochrome c with Anionic Surfactants

The kinetics of absorbance and fluorescence changes of cytochrome c as induced by an aqueous solution of the anionic surfactant sodium dodecyl sulfate (SDS) or sodium bis(2-ethylhexyl)sulfosuccinate (AOT) are studied. The results are compared with far-UV circular dichroism (CD) spectra. Both surfactants cause similar alterations in the secondary structure of cytochrome c, while their influence on the heme environment of cytochrome c is different. In the presence of AOT below and above critical micellar concentration a conversion of the low-spin native cytochrome c to a denatured low-spin protein not having methionine ligand takes place. In the presence of SDS micelles conversion of the native protein to a denatured mixed-spin form occurs. The changes in the heme group induced by both surfactants occur independently of the alterations in tertiary structure.     

Crucial role of the lipid part of lipopolysaccharide for conformational change of minor spike H protein of bacteriophage phiX174

The contribution of the lipid part of lipopolysaccharide (LPS) to recognition by minor spike H protein of bacteriophage phiX174 was investigated by comparing the interactions of H protein with LPS and its deacylated derivatives. The fluorescence and circular dichroism (CD) spectra of H protein increased upon binding to intact LPS and a partially deacylated derivative. In contrast, completely deacylated derivatives showed lower affinities and almost no fluorescence or CD changes of H protein. These results demonstrate that the lipid part of LPS is responsible for the conformational change of minor spike H protein, which would function as a trigger for phage DNA ejection for infection of the host cell.     

The non-native conformations of cytochrome c in sodium dodecyl sulfate and their modulation by ATP

To understand the interaction of cytochrome c (cyt c) with membranes, a systematic investigation of sodium dodecyl sulfate (SDS)-induced conformational alterations in native horse heart ferricytochrome c (pH 7.0) was carried out using heme absorbance, tryptophan fluorescence and circular dichroism (CD) spectroscopy. ATP interaction with membrane-bound cyt c is known to regulate the process of apoptosis. To understand the effect of nucleotide phosphates on membrane-bound cyt c, we also carried out studies of the interaction of ATP with cyt c in the presence of SDS. Fluorescence and UV-Vis data suggest that SDS induces two different transitions (F to C1, C1 to C2) in cyt c, one in the pre-micellar region and the other in the post-micellar region. The fluorescence data further indicated the increase in distance between Trp 59 and heme in the intermediates in both the regions, suggesting loosening up of cyt c on titration with SDS. The far-UV and near-UV CD data suggest partial loss of secondary and tertiary structure in C1, but complete loss of tertiary structure and no further loss of secondary structure in C2. On titration of C1 and C2 with ATP, the secondary structure is restored. However, the heme ligation pattern and heme exposure change only for C2, but not for C1 on the addition of ATP.     

Conformational changes of beta-lactoglobulin induced by anionic phospholipid

Conformational changes of beta-lactoglobulin (beta-LG) induced by anionic phospholipid (dimyristoylphosphatidylglycerol, DMPG) at physiological conditions (pH 7.0) have been investigated by UV-VIS, circular dichroism (CD) and fluorescence spectra. The experimental results suggest that beta-LG-DMPG interactions cause beta-LG a structural reorganization of the secondary structure elements accompanied by an increase in alpha-helical content, and a loosening of the protein tertiary structure. The interaction forces between beta-LG and DMPG are further evaluated by fluorescence spectra. The fluorescence spectral data show that conformational changes in the protein are driven by electrostatic interaction at first, then by hydrophobic interaction between a protein with a negative net charge and a negatively charged phospholipid.     

Investigation on the Interaction of Hydroxyethyl Starch 130/0.4 (Voluven) and Serum Albumin for Pharmacokinetic and Toxicological Implications

The interaction of hydroxyethyl starch 130/0.4 (Voluven) with human serum albumin (HSA) has been investigated by fluorescence (steady state and synchronous), Fourier transforms infrared (FT‐IR), and circular dichroism (CD) spectroscopies. Analysis of the fluorescence quenching data of HSA by Voluven using the Stern–Volmer method revealed the formation of 1:1 ground‐state complex. Evaluation of binding parameters and binding energy indicated that the binding reaction was exothermic. On the basis of fluorescence measurements, it was concluded that electrostatic forces play a crucial role in stabilizing the complex. The binding distance was calculated by using Förster resonance energy transfer (FRET) theory. The conformational changes of HSA were obtained qualitatively as well as quantitatively using synchronous fluorescence, FT‐IR, and CD. The HSA underwent partial unfolding in the presence of Voluven.     

Optical spectroscopy to investigate the structure of regenerated Bombyx mori silk fibroin in solution

Fluorescence and circular dichroism spectroscopy were used to monitor the conformational transition of regenerated Bombyx mori silk fibroin (RSF) in aqueous solutions under different conditions. According to the analysis of fluorescence spectra using anilinonaphthalene-8-sulfonic acid magnesium salt (ANS) as an external probe, the destruction of the hydrophobic core prior to the secondary structure change suggests that this collapse may initiate the conformational transition from random coil to beta-sheet for RSF. The temperature dependence of the structural changes of RSF, detected by both fluorescence spectroscopy and circular dichroism, shows a reversible process upon heating and recooling, with the midpoint around 45 degrees C. The results also indicate that most of the tryptophan (Trp) residues contained in silk fibroin are concentrated on the surface of the unfolded protein. However, they will change their location in the highly ordered structure (e.g., becoming more homogeneous) with the conformational transition of silk fibroin. Moreover, our studies also suggest that the presence of water plays a crucial role during the structure changes of fibroin.     

Investigation of binding properties of dicationic styrylimidazo[1,2‐a]pyridinium dyes to human serum albumin by spectroscopic techniques

The binding interaction between two dicationic styrylimidazo[1,2‐a]pyridinium dyes and human serum albumin (HSA) was investigated at physiological conditions using fluorescence, UV–vis absorption, and circular dichroism (CD) spectroscopies. Analysis of the fluorescence titration data at different temperatures suggested that the fluorescence quenching mechanism of HSA by these dyes was static. The calculated thermodynamic parameters (ΔG°, ΔH° and ΔS°) indicated that hydrogen bonding and van der Waals forces played a major role in the formation of the dye–HSA complex. Binding distances (r) between dyes and HSA were calculated according to Förster's non‐radiative energy transfer theory. Studies of conformational changes of HSA using CD measurements indicate that the α‐helical content of the protein decreased upon binding of the dyes. Copyright © 2016 John Wiley & Sons, Ltd.     

Effects of trehalose and sorbitol on the activity and structure of Pseudomonas cepacia lipase: spectroscopic insight

The stability of enzymes with no reduction in their catalytic activity still remains a critical issue in industrial applications. Naturally occurring osmolytes are commonly used as protein stabilizers. In this study we have investigated the effects of sorbitol and trehalose on the structural stability and activity of Pseudomonas cepacia lipase (PCL), using UV-visible, circular dichroism (CD) and fluorescence spectroscopy. Surface plasmon resonance (SPR) technique was used to trace changes in the refractive index and dielectric constant of the environment. The results revealed that catalytic activity and intrinsic fluorescence intensity of PCL increased in the presence of both osmolytes. Far-UV CD spectra indicated that the protein has undergone some conformational changes upon interacting with these osmolytes. Increasing the concentration of sorbitol led to changes in the refractive index and consequently the dielectric constant of environment; whereas in the case of trehalose, such changes were not significant. Unfavorable interactions of trehalose with protein surface induced higher preferential exclusion from the enzyme-water interface than that of sorbitol. Results of this report could give further insights about the stabilization mechanism of osmolytes.     

Interactions between antimalarial indolone-N-oxide derivatives and human serum albumin

The binding affinity of human serum albumin (HSA) to three antimalarial indolone-N-oxide derivatives, INODs, was investigated under simulated physiological conditions using fluorescence spectroscopy in combination with UV-vis absorption and circular dichroism (CD) spectroscopy. Analysis of fluorescence quenching data of HSA by these compounds at different temperatures using Stern-Volmer and Lineweaver-Burk methods revealed the formation of a ground state indolone-HSA complex with binding affinities of the order 10(4) M(-1). The thermodynamic parameters ΔG, ΔH, and ΔS, calculated at different temperatures, indicated that the binding reaction was endothermic and hydrophobic interactions play a major role in this association. The conformational changes of HSA were investigated qualitatively using synchronous fluorescence and quantitatively using CD. Site marker competitive experiments showed that the binding process took place primarily at site I (subdomain IIA) of HSA. The number of binding sites and the apparent binding constants were also studied in the presence of different ions.     

Comparison of binding interactions of lomefloxacin to serum albumin and serum transferrin by resonance light scattering and fluorescence quenching methods

The interaction between lomefloxacin (LMF) and two drug carrier proteins, human serum albumin (HSA) and serum transferrin (TF), were studied and compared by fluorescence quenching, resonance light scattering (RLS), and circular dichroism (CD) spectroscopic along with molecular modeling. Fluorescence data show that LMF has a stronger quenching effect on HSA than on TF. The binding constant and the number of binding sites were calculated as 6.00 x 10(5) M(-1) and 0.77 for HSA, and 4.66 x 10(5) M(-1) and 1.02, for TF, respectively. Also, these binding parameters were calculated by RLS data, as a novel approach and were compared to that obtained from fluorescence. The micro-environment changes of Trp residues were evident in both proteins. The quantitative analysis of the secondary structure in both proteins further confirmed the drug-induced conformational changes. The distance (r) between donors (HSA and TF) and acceptor (LMF) were obtained by fluorescence resonance energy transfer (FRET) theory and found to be 1.83 nm and 1.71 nm for HSA and TF respectively. Moreover, molecular modeling studies suggested the sub-domain IB in HSA and N-lobe in TF as the candidate place for the formation of the binding site of LMF on these proteins.