Protein-surfactant Interactions Research Articles

Polymerized whey protein-SDS interactions at their high concentrations

Protein-surfactant interactions have been an ongoing topic of interest for many decades. Applications involving complexes of proteins and surfactants are relevant in food, pharmaceuticals, hygiene, molecular characterization, and other fields. In this study, the interactions of polymerized whey proteins (PWP) and sodium dodecyl sulfate (SDS) at high concentrations are investigated. Different characterization techniques are used, including electrical conductivity, turbidity, isothermal titration calorimetry, dynamic light scattering, dilute solution viscometry, rheology, and surface hydrophobicity to elucidate information on the modes and extent of interactions. Results indicate that PWP-SDS interactions produce highly extended, worm like micelles, with SDS decorating PWP chains and covering non-polar residues. PWP can host SDS up to quite high surfactant to protein ratios (SPR), producing solutions that are highly viscous with shear thickening properties, yet with no networking or gelation. Interestingly, dilution of high viscosity PWP-SDS solutions leads to even smaller size of PWP-SDS molecular complex as compared with PWP without SDS. The current study extends the vision of protein surfactant interactions by examining concentration range beyond that found in literature. The results reveal insights that can help expand studies on other systems and find applications in various fields including coatings, cosmetics, food ingredients, drug transport, and disease treatment.

Impedance Spectroscopy Unveiled the Surfactant-Induced Unfolding and Subsequent Refolding of Human Serum Albumin.

Protein-surfactant interaction is a dynamic interplay of electrostatic and hydrophobic forces that ensues from the folding of a protein. We employ impedance spectroscopy (IS), a label-free method, to investigate the unfolding and refolding of human serum albumin (HSA), a globular plasma protein, in the presence of two surfactants: polysorbate-20 (Tween-20), a nonionic surfactant, and sodium dodecyl sulfate (SDS), an anionic surfactant. The equivalent electrical analog circuit was predicted from impedance spectra of HSA in an aqueous solution at physiological pH and room temperature, focusing on varying the concentration of codissolved surfactants. A change in the dielectric constant (ε') and ionic conductivity (κ) is observed by comparing the surfactant-treated protein samples to the bare surfactant solutions to assess the conformational changes induced by surfactants in HSA. Far-UV circular dichroism analysis revealed a decrease in α-helices and an increase in β-sheets and random coils upon SDS addition, which were reversed by Tween-20. Dynamic light scattering supported the findings by measuring changes in the hydrodynamic diameter (dh) of HSA. Unfolding and refolding of HSA with surfactants were also observed through photoluminescence spectroscopy by examining the microenvironment surrounding the single tryptophan (W) within the protein, and the thermodynamic parameters were obtained using the modified Stern-Volmer equation. Our research explores the intriguing domain of protein-surfactant interactions, offering insights with promising applications across diverse biological processes and IS as a suitable alternative technique for investigating protein conformational changes by studying the electrical response of the samples.

Physicochemical evaluation of interaction behavior of a series of biocompatible gemini surfactants with hemoglobin: Insights from spectroscopic and computational studies

In this study, we investigated the interaction behavior of biodegradable, cationic, oxy-diester functionalized gemini surfactants N1,N1,N14,N14-tetramethyl-2,13-dioxo-N1,N14-dialkyl-3,6,12-tetraoxateradecane-1,14-diaminium dichloride (in short Cm-E2O2-Cm, m = 12, 14 or 16 is the number of carbon atoms in the alkyl chain) with one of the most important proteins in red blood cells viz. hemoglobin. We employed a variety of in-vitro and in-silico methodologies, such as fluorescence, UV–vis, FT-IR and circular dichroism spectroscopies, DFT, molecular docking, and molecular dynamics simulations, to study these interactions. Our results show that the chain length of the surfactants have distinct implications for the interaction behavior, as evidenced by the estimated physical parameters, such as Kb and ∆G. The order of Kb values (0.24 × 103 < 3.11 ×103 < 5.85 × 103 M-1) is in accordance with the increasing alkyl chain length of the surfactants. The negative Gibbs free energy change values and their order, − 13.58 < −19.93 < −21.49, further support the spontaneity of interactions and the effect of alkyl chain length. Theoretical insight into the environment at the quantum mechanical level, binding site, and energy stability of the protein-surfactant interactions were produced by computational methods. This research contributes significantly to the field of knowledge concerning surfactants' effects on protein structure and behavior.

Molecular interaction of di-ester bonded cationic Gemini surfactants with pepsin: in vitro and in silico perspectives

The implications of surfactant–enzyme/protein interactions in a variety of fields, including biotechnology, cosmetics, paints and pharmaceuticals, have attracted a lot of attention in contemporary studies. Herein, we have employed several in vitro and in silico techniques such as excitation and absorption spectroscopies, circular dichroism and FT-IR spectroscopies, density functional and molecular dynamics simulations to understand the interaction behavior of oxy-diester-based green cationic Gemini surfactants, N1,N1,N14,N14-tetramethyl-2,13-dioxo-N1,N14-dialkyl-3,6,12-tetraoxateradecane-1,14-diaminiumdichloride (abbreviated as Cm-E2O2-Cm, where ‘m’ stands for alkyl chain length, m = 12 and 14) with one of the main digestive proteins, pepsin. The spectroscopic techniques confirm the static quenching effect of surfactants on pepsin. The calculated physical parameters (Ksv , Kb and ΔG) and their order reveal the distinguished implications for the surfactants’ chain lengths. The spontaneity of interaction was also confirmed by negative Gibbs free energy change values. The extrinsic spectroscopic study with pyrene as fluorescence probe, FT-IR and CD techniques indicated a potential conformational change in pepsin induced by the Gemini surfactants. DFT, docking and MD simulations provided the theoretical understanding regarding the quantum mechanical environment, location of binding and stability of the protein–surfactant complexation in energy terms. We believe this study will be a humble addition to our existing knowledge in the field of protein–surfactant interactions. Communicated by Ramaswamy H. Sarma

Interactions between Mannosylerythritol Lipid-A and Heat-Induced Soy Glycinin Aggregates: Physical and Chemical Characteristics, Functional Properties, and Structural Effects.

Protein-surfactant interactions have a significant influence on food functionality, which has attracted increasing attention. Herein, the effect of glycolipid mannosylerythritol lipid-A (MEL-A) on the heat-induced soy glycinin (11S) aggregates was investigated by measuring the structure, binding properties, interfacial behaviors, and emulsification characteristics of the aggregates. The results showed that MEL-A led to a decrease in the surface tension, viscoelasticity, and foaming ability of the 11S aggregates. In addition, MEL-A with a concentration above critical micelle concentration (CMC) reduced the random aggregation of 11S protein after heat treatment, thus facilitating the formation of self-assembling core-shell particles composed of a core of 11S aggregates covered by MEL-A shells. Infrared spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry also confirmed that the interaction forces between MEL-A and 11S were driven by hydrophobic interactions between the exposed hydrophobic groups of the protein and the fatty acid chains or acetyl groups of MEL-A, as well as the hydrogen bonding between mannosyl-D-erythritol groups of MEL-A and amino acids of 11S. The findings of this study indicated that such molecular interactions are responsible for the change in surface behavior and the enhancement of foaming stability and emulsifying property of 11S aggregates upon heat treatment.

Insights into the Stabilization of Interferon Alpha by Two Surfactants Revealed by STD-NMR Spectroscopy

Surfactants are commonly used in biopharmaceutical formulations to stabilize proteins against aggregation. However, the choice of a suitable surfactant for a particular protein is decided mostly empirically, and their mechanism of action on molecular level is largely unknown. Here we show that a straightforward label-free method, saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, can be used to detect protein-surfactant interactions in formulations of a model protein, interferon alpha. We find that polysorbate 20 binds with its fatty acid to interferon, and that the binding is stronger at pH closer to the isoelectric point of the protein. In contrast, we did not detect interactions between poloxamer 407 and interferon alpha. Neither of the two surfactants affected the tertiary structure and the thermal stability of the protein as evident from circular dichroism and nanoDSF measurements. Interestingly, both surfactants inhibited the formation of subvisible particles during long-term storage, but only polysorbate 20 reduced the amount of small soluble aggregates detected by size-exclusion chromatography. This proof-of-principle study demonstrates how STD-NMR can be employed to quickly assess surfactant-protein interactions and support the choice of surfactant in protein formulation.

Surfactant-Assisted Assembly of Dipeptide Forming a Broom-like Structure.

Understanding the influence of surfactants on the assembly of peptides has a considerable practical motivation. In this paper, we systematically study the anionic surfactant-assisted assembly of diphenylalanine (FF). FF forms broom-like structures in a concentration of sodium cholate (NaC) around the CMC, and assembles into linear and unidirectional rods in the presence of low and high surfactant concentrations. FF’s improved hydrogen bonding and controlled assembly rates are appropriate for other anionic surfactants. At this stage, the use of FF as the simplest protein consequence can be helpful in the investigation of further protein–surfactant interactions.

Deciphering the mechanism of interaction of an ester-functionalized cationic gemini surfactant with bovine serum albumin: A biophysical and molecular modeling study

Protein–surfactant interactions have relevance in the fields of pharmaceuticals, detergency formulation, pharmacology, personal hygiene products, etc. Accordingly, we have analyzed the biomolecular interaction between a recently synthesized C16-E2O2-C16 gemini surfactant with the transport protein, bovine serum albumin (BSA), utilizing surface tension, fluorescence (intrinsic/extrinsic, synchronous, 3-D and resonance Rayleigh scattering (RRS)), circular dichroism (CD), UV–visible, Fourier transform infrared (FT-IR) and computational methods. The critical micelle concentration (CMC) and surface parameters of the gemini surfactant get modified in the presence of BSA. The interaction of the concerned surfactant with BSA occurred via a static quenching mechanism forming a less stable complex at the higher temperature. Based on calculated thermodynamic parameters, the interaction process was found to be spontaneous (∆Gb0 is negative) and that hydrogen bond (HB) and van der Waals (VDW) forces were predominant forces in stabilizing the complex (both ∆Hb0 and ∆Sb0 are negative). FT-IR and CD measurements demonstrated that the secondary structure conformation of BSA was perturbed because of the BSA-C16-E2O2-C16 complexation. The 3-D and RRS fluorescence studies demonstrated that the BSA size enlarged in presence of the gemini and ensued in the increment of scattering effect and suggested some conformational and micro-environmental alterations of Trp/Tyr residues of BSA (the same results were obtained for pyrene extrinsic fluorescence). Molecular docking and site marker displacement investigations recognized that the surfactant C16-E2O2-C16 predominantly binds within BSA’s site I (sub-domain IIA).

On the Effect of pH, Temperature, and Surfactant Structure on Bovine Serum Albumin-Cationic/Anionic/Nonionic Surfactants Interactions in Cacodylate Buffer-Fluorescence Quenching Studies Supported by UV Spectrophotometry and CD Spectroscopy.

Due to the fact that surfactant molecules are known to alter the structure (and consequently the function) of a protein, protein–surfactant interactions are very important in the biological, pharmaceutical, and cosmetic industries. Although there are numerous studies on the interactions of albumins with surfactants, the investigations are often performed at fixed environmental conditions and limited to separate surface-active agents and consequently do not present an appropriate comparison between their different types and structures. In the present paper, the interactions between selected cationic, anionic, and nonionic surfactants, namely hexadecylpyridinium chloride (CPC), hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), polyethylene glycol sorbitan monolaurate, monopalmitate, and monooleate (TWEEN 20, TWEEN 40, and TWEEN 80, respectively) with bovine serum albumin (BSA) were studied qualitatively and quantitatively in an aqueous solution (10 mM cacodylate buffer; pH 5.0 and 7.0) by steady-state fluorescence spectroscopy supported by UV spectrophotometry and CD spectroscopy. Since in the case of all studied systems, the fluorescence intensity of BSA decreased regularly and significantly under the action of the surfactants added, the fluorescence quenching mechanism was analyzed thoroughly with the use of the Stern–Volmer equation (and its modification) and attributed to the formation of BSA–surfactant complexes. The binding efficiency and mode of interactions were evaluated among others by the determination, comparison, and discussion of the values of binding (association) constants of the newly formed complexes and the corresponding thermodynamic parameters (ΔG, ΔH, ΔS). Furthermore, the influence of the structure of the chosen surfactants (charge of hydrophilic head and length of hydrophobic chain) as well as different environmental conditions (pH, temperature) on the binding mode and the strength of the interaction has been investigated and elucidated.

Comparative scrutinize of BSA and HEWL in the vicinity of metallo-catanionic aggregates derived from single chain metallosurfactant and anionic surfactant

In recent times, protein-surfactant interactions have been extensively explored due to their stimulating applications in different fields. These systems have efficaciously addressed the economic, environmental, and ecological issues. Sensing their prominence, through this report, we investigate and compare the interactional behavior of Bovine Serum Albumin (BSA) and Hen Egg White Lysozyme (HEWL) with metallo-catanionic aggregate mixtures. Attempts have been made to synthesize the metallo-catanionic aggregates from single-chain metallosurfactant (FeCTAC I) and anionic surfactant (SDS) i.e., FeCTAC I:SDS. Before proceeding towards the interactional studies of metallo-catanionic aggregates with model proteins, the morphology of formed aggregates was elucidated. Here, out of different possible molar ratios of FeCTAC I:SDS, two ratios i.e 70:30 and 30:70 were opted. The aggregates were examined through conductivity, Dynamic Light Scattering (DLS), Field Emission Scanning Electron Microscopy (FESEM), Confocal Laser Scanning Microscopy (CLSM), Small Angle X-Ray Scattering (SAXS), Zeta Potential, and Vibrating Sample Magnetometer (VSM) techniques. Interestingly, the SAXS analysis demonstrated the micellar behavior of 70:30 and vesicular behavior of 30:70 aggregates mixture, due to the difference of overall charge on them. The interactional study of BSA and HEWL with FeCTAC I:SDS was carried out through SDS-PAGE, absorption, emission, and circular dichroism (CD) spectroscopy. The effect of FeCTAC I:SDS on the linear polypeptide chain of BSA and HEWL was observed to be different, however, the secondary and tertiary structures were affected similarly. To elucidate the exact locality of FeCTAC I:SDS aggregates on the BSA, a site marker experiment was carried out. The pyrene extrinsic fluorescence experiment was performed to verify the type of interactions among HEWL and metallo-catanionic aggregates. From, the comparative studies, we concluded that the overall system was controlled by the type of forces playing between the protein and aggregate mixture that emerged from the total charge on the protein as well as on the aggregates. These types of metallo-catanionic aggregates that are flexible to shape are considered as the new alternatives for nano-vehicle applications.

Semi-empirical Analysis of Complex ITC Data from Protein-Surfactant Interactions.

Isothermal titration calorimetry (ITC) is a widely used method to determine binding affinities and thermodynamics in ligand-receptor interactions, but it also has the capability of providing detailed information on much more complex events. However, the lack of available methods to analyze ITC data is limiting the use of the technique in such multifaceted cases. Here, we present the software ANISPROU. Through a semi-empirical approach that allows for extraction of quantitative information from complex ITC data, ANISPROU solves an inverse problem where three parameters describing a set of predefined functions must be found. In analogy to strategies adopted in other scientific fields, such as geophysics, imaging, and many others, it employs an optimization algorithm which minimizes the difference between calculated and experimental data. In contrast to the existing methods, ANISPROU provides automated and objective analysis of ITC data on sodium dodecyl sulfate (SDS)-induced protein unfolding, and in addition, more information can be extracted from the data. Here, data series on SDS-mediated protein unfolding is analyzed, and binding isotherms and thermodynamic information on the unfolding events are extracted. The obtained binding isotherms as well as the enthalpy of different events are similar to those obtained using the existing manual methods, but our methodology ensures a more robust result, as the entire data set is used instead of single data points. We foresee that ANISPROU will be useful in other cases with complex enthalpograms, for example, in cases with coupled interactions in biomolecular, polymeric, and amphiphilic systems including cases where both structural changes and interactions occur simultaneously.

Current trends in protein-surfactant interactions: A review

The protein-surfactant interactions have been investigated for a long time due to their increased effectiveness in cleanliness and industrial processes. Moreover, Covid-19 outbreak has revolutionized the significance of protein-surfactant interactions, as soaps were proved the best cleansers and safe during this period. In this review article, an overview of physico-chemical aspects of protein-surfactant interactions along with various techniques has been discussed. In addition, the effect of different factors such as co-solvent, temperature, pH, chain length of surfactants, salt, and their impact on critical micelle concentration (CMC) of surfactant, structure, binding, thermodynamic parameters, and stability was also explained. This review will help the scientific community in understanding how to improve the effectiveness and applications of proteins and surfactants in pharmaceutical and other industries as protein-surfactant interactions can explain the significant phenomena of hydrophobic and electrostatic interactions.

Centrosomal Localization of RXRα Promotes PLK1 Activation and Mitotic Progression and Constitutes a Tumor Vulnerability.

Retinoid X receptor alpha (RXRα), a nuclear receptor of transcription factor, controls various physiological and pathological pathways including cellular growth, proliferation, differentiation, and apoptosis. Here, we report that RXRα is phosphorylated at its N-terminal A/B domain by cyclin-dependent kinase 1 (Cdk1) at the onset of mitosis, triggering its translocation to the centrosome, where phosphorylated-RXRα (p-RXRα) interacts with polo-like kinase 1 (PLK1) through its N-terminal A/B domain by a unique mechanism. The interaction promotes PLK1 activation, centrosome maturation, and mitotic progression. Levels of p-RXRα are abnormally elevated in cancer cell lines, during carcinogenesis in animals, and in clinical tumor tissues. An RXRα ligand XS060, which specifically inhibits p-RXRα/PLK1 interaction but not RXRα heterodimerization, promotes mitotic arrest and catastrophe in a tumor-specific manner. These findings unravel a transcription-independent action of RXRα at the centrosome during mitosis and identify p-RXRα as a tumor-specific vulnerability for developing mitotic drugs with improved therapeutic index.

Binding isotherms of surfactants used in detergent formulations to bovine serum albumin

Protein-surfactant interactions are the focus of extensive research due to their many applications in food technology and detergent industry. In this work, we investigate the interaction between bovine serum albumin (BSA) and five relevant surfactants to the cleaning industry, which differ in head group charge, namely: sodium alkyl ether sulphate (C12-C14 AE3S), Cocoamidopropyl amine-oxide (CapAO), alkyl dimethyl amine oxide (C12C14AO), octaethylene glycol monodecyl ether (C10EO8) and didecyldimethylammonium chloride (DDAC). The results collected with fluorescence emission spectroscopy highlight zwitterionic and nonionic surfactants have the lowest affinity for the protein, as their interaction does not result in protein denaturation. Instead, higher and mutually close binding constants are found for AE3S (anionic) and DDAC (cationic) due to the presence of electrostatic interactions between the surfactant heads and the charged residues of BSA. AE3S leads to irreversible protein unfolding. The case of DDAC is more complex and has been studied through a combination of fluorescence, DLS, PGSE-NMR and zeta-potential measurements. At low concentration DDAC binding neutralizes negatively charged residues present in BSA, causing a reversible flocculation of BSA after the isoelectric point (23 DDAC molecules per protein). Further DDAC adsorption, at 70 DDAC molecules per protein, leads to an excess of positive charge on the protein which restores electrostatic repulsions between BSA-surfactant complexes.

Development of Non-ionic Surfactant and Protein-Coated Ultrasmall Silver Nanoparticles: Increased Viscoelasticity Enables Potency in Biological Applications.

To enhance the interactivity with biological cells, we developed ultrasmall (5 nm in diameter) Ag NPs coated with a mixture of Tween-20 (Tw-20) surfactant and human serum albumin (HSA) or hemoglobin (Hb) proteins. These were tested with cancerous and healthy cell lines to investigate the therapeutic applicability. Using the established concept of generation of reactive oxygen species (ROS) and the ROS-induced oxidative stress in carcinogenic cells by Ag NPs, we found that the presently synthesized Ag NPs selectively destroyed the cancerous cells. A mixture of Tw-20 with protein, where the surfactant was in large excess, created a coating over the Ag NPs resulting weaker protein–protein interactions and facilitating interfacial protein–surfactant interactions, which leads to an increase in the film viscoelasticity to enhance the stability of the Ag NPs and cell viability. Moreover, this concept has been applied to drug delivery using a model fluorophore (fluorescein) on Ag NPs to explore the prospects in photodynamic therapy. The results are encouraging and deserve further investigation.

Protein-surfactant interactions: A multitechnique approach on the effect of Co-solvents over bovine serum albumin (BSA)-cetyl pyridinium chloride (CPC) system

In this work, thermodynamic parameters of micellization and binding of protein with surfactant in presence of co-solvents were derived using experimental and theoretical methods. Multi Ligand Simultaneous Docking (MLSD) was done with 8 pocket sites of BSA and BP3 behaved as the best binding site for CPC + Glycerol and CPC + DMSO. Finally, quantum chemistry calculations showed that the main intermolecular interactions between the (CPC + Glycerol)-BSA and (CPC + DMSO)-BSA were mainly governed by hydrogen bonding and van der Waals forces, promoting micelle formation and allowing the supramolecular assemblies. The theoretical calculations are in good agreement with the experimental results.

A physicochemical and conformational study of co-solvent effect on the molecular interactions between similarly charged protein surfactant (BSA-SDBS) system

Use of surfactants in numerous household and industrial processes and their interaction with proteins in our day to day life has made protein-surfactant interactions a booming topic among the researchers of current era. Bovine serum albumin (BSA) being structural homologue to Human Serum albumin (HSA) allowed us to study its binding efficiency with anionic surfactant such as sodium dodecyl benzene sulfonate (SDBS). BSA is known as transport protein due its binding characteristics as well as transportation of hydrophobic drugs or ligands to different target areas (In Human body). Surfactants are also known as eminent constituents of pharmaceutical drug delivery systems. So, interactions of BSA-SDBS (Similarly charged protein-surfactant system) in presence of two industrially important co-solvents DMSO and Glycerol have decided its fate as a novel drug delivery system. These interactional studies were performed by means of myriad experimental and theoretical approaches i.e. conductivity, fluorescence, Ligand Simultaneous Docking (MLSD) and non-covalent interactions index (NCI). Results showed the dominance of Van der Waals interactions and hydrophobic forces with important changes in the preferred binding site.

Pre-micellar concentrations of sodium dodecylbenzene sulphonate induce amyloid-like fibril formation in myoglobin at pH 4.5

Although many reports have described protein–surfactant interactions but the mechanism of amyloid-like fibril formation by surfactants is not clear. In this study, we investigated the role of sodium dodecylbenzene sulfonate (SDBS) to induce aggregation in equine heart muscle myoglobin (E–Mb) at pH 4.5. The aggregation was analyzed by Rayleigh scattering (RLS), turbidity, Thioflavin-T binding (ThT), and Congo red (CR) binding, circular dichroism spectroscopy and transmission electron microscopy (TEM). Turbidity and RLS analysis demarcated the aggregate were formed at effective range of SDBS concentration at pH 4.5, this led to the observation that at low concentrations (0.05 mM–1.2 mM) SDBS cause aggregation of E–Mb monomers, in contrast at higher SDBS concentrations the aggregates were not formed. The following observations were further supported through ThT and CR binding results suggests that SDBS-induced E–Mb aggregates have amyloid-like structure. Far-UV CD results for aggregated E–Mb also displayed single negative peak between 222−235 nm, which is a characteristic of cross-β-sheet. However, in the presence of high concentration of SDBS, the non-aggregated state of E–Mb obtained consisted of mixed α-helices and β-sheets. SDBS-induced aggregates had amyloid-like morphology as confirmed by TEM. These results indicate that both electrostatic forces and hydrophobic interactions contribute towards SDBS-induced E–Mb aggregates formation with a characteristic amyloid-like structure. Also, these findings have significant implications for understanding mechanisms underlying amyloid fibril formation by anionic surfactants.

Unfolding and partial refolding of a cellulase from the SDS-denatured state: From β-sheet to α-helix and back

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C12E8). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C12E8 leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C12E8 were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3–4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C12E8 recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C12E8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.

Myoglobin and α-Lactalbumin Form Smaller Complexes with the Biosurfactant Rhamnolipid Than with SDS

Biosurfactants (BSs) attract increasing attention as sustainable alternatives to petroleum-derived surfactants. This necessitates structural insight into how BSs interact with proteins encountered by current chemical surfactants. Thus, small-angle x-ray scattering (SAXS) has been used for studying the structures of complexes made of the proteins α-Lactalbumin (αLA) and myoglobin (Mb) with the biosurfactant rhamnolipid (RL). For comparison, complexes between αLA and the chemical surfactant sodium dodecyl sulfate (SDS) were also investigated. The SAXS data for pure RL micelles can be described by prolate core-shell structures with a core radius of 7.7 Å and a shell thickness of 12 Å, giving an aggregation number of 11. The small core radius is attributed to RL’s complex hydrophobic tail. Data for the αLA-RL complex agree with a 12-molecule micelle with a single protein molecule in the shell. For Mb-RL, the analysis gives complexes of two connected micelles, each containing 10 RL and one protein in the shells. αLA-RL and Mb-RL form surfactant-saturated complexes above 5.6 and 4.7 mM RL, respectively, leaving the remaining RL in free micelles. The SAXS data for SDS agree with oblate-shaped micelles with a core of 20 Å, core eccentricity 0.7, and shell thickness of 5.45 Å, with an aggregation number of 74. The αLA-SDS complexes contain a prolate micelle with a core radius of 11−14 Å and a shell of 8−12 Å with up to 3 αLA per particle and up to 43 SDS per αLA, both considerably larger than for RL. Unlike the RL-protein complexes, the number of surfactant molecules in αLA-SDS complexes increases with surfactant concentration, and saturate at higher surfactant concentrations than αLA-RL complexes. The results highlight how RL and SDS follow similar overall rules of self-assembly and interactions with proteins, but that differences in the strength of protein-surfactant interactions affect the formed structures.