Sodium Dodecyl Sulfate Denaturation Research Articles

Effects of Different Denaturants on the Properties of a Hot-Pressed Peanut Meal-Based Adhesive.

Plant protein-based adhesives could fundamentally solve the problem of formaldehyde-based adhesive releasing formaldehyde, but enhancing bonding strength and water resistance is a necessary measure to realize practical applications. In this study, the effects of different denaturants on the properties of a hot-pressed peanut meal (HPM)-based adhesive before and after crosslinking were studied. Papain, sodium dodecyl sulfate (SDS), urea and crosslinker-polyamide epichlorohydrin (PAE) were used to prepare HPM-based adhesives. The functional groups, bonding strength, thermal behaviors, mass loss, moisture uptake value, viscosity and fracture surface of adhesive samples were analyzed. As a result, (1) papain was used to break HPM protein (HPMP) into polypeptide chains and to reduce the water resistance. (2) SDS and urea unfold the HPMP molecule and expose internal hydrophobic groups to improve the water resistance of the adhesive. (3) A denser network structure was formed by PAE and HPMP molecules, which significantly improved the bonding strength and water resistance of adhesives. In particular, after SDS denaturation and PAE crosslinking, compared with pure HPM adhesive, the wet shear strength increased by 96.4%, the mass loss and moisture uptake value reduced by 41.4% and 69.4%, and viscosity increased by 30.4%. This work provided an essential guide to design and prepare HPM-based adhesives.

Physicochemical characterization and linear epitopes identification of arginine kinase allergen from Crassostrea gigas.

Molluscan shellfish, including oysters, often cause allergic reactions in sensitive people throughout the world. It has been demonstrated that arginine kinase (AK) is one of the major allergens of oyster. The present study aimed to evaluate the immunoreactivity and structure of oyster AK as affected by heat treatment, pH change, and in vitro digestion. What is more, the immunoglobulin E-binding epitopes of this allergen were also predicted and validated. Thermal and pH assays revealed that AK was unstable at temperature >40 °C or pH ≤5.0 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and circular dichroism, and the digestibility assays suggested that AK was more easily digested by pepsin than by trypsin and chymotrypsin. The potential epitopes were predicted through immunoinformatics tools, and seven linear epitopes were identified by indirect competition enzyme-linked immunosorbent assay with pooled sera and individual serum from oyster-allergic patients. The critical amino acids in each epitope were also confirmed using mutant peptides. These linear epitopes and critical amino acids were apt to distribute on the outer surface of homology-based AK model. Moreover, the three denaturants (sodium dodecyl sulfate, β-mercaptoethanol, and urea) can destroy the spatial structure of AK and increase or reduce its allergenicity by denaturation treatments. Processing conditions lay the foundation for the variation of allergenicity. Seven linear epitopes and their critical amino acids were identified by indirect competitive enzyme-linked immunosorbent assay. These findings will be helpful in allergy diagnosis and development of hypoallergenic products in the near future. © 2021 Society of Chemical Industry.

A novel environmentally friendly hot-pressed peanut meal protein adhesive

The plant protein-based adhesives are a promising alternative to petroleum-based adhesives, while the low bonding strength, water resistance and mildew-proofing limit their application in plywood industry. In this study, hot-pressed peanut meal protein (HPMP) adhesive with enhanced bonding strength was successfully prepared using a three-step method of sodium dodecyl sulfate (SDS) denaturation, nano-silica (nSiO2) reinforcement and polyamide polyamine epichlorohydrin resin (PAE) crosslinking. The boiling water strength of HPMP adhesive (0.85 MPa) exceeded the bonding strength requirement of China National Standards class Ⅰ plywood (0.7 MPa), and the mildew resistance of HPMP adhesive was extended at least 12 days. Simultaneously, the solids content and viscosity of HPMP adhesive increased by 45.4% and 274.6% respectively. The bonding strength-enhancing mechanism of HPMP adhesive was further clarified by using HPM protein isolate (HPMPI) adhesive. That is, the ring network structures were formed by connecting with hydrogen bonds, ester bonds and ether bonds, and clearly observed by double spherical aberration-corrected scanning transmission electron microscopy. Thus, the prepared novel HPMP adhesive has great potential for application as an environmental-friendly commercial wood adhesive for plywood production.

Optimization and practical application of cottonseed meal-based wood adhesive formulations for small wood item bonding

The interest in biobased wood adhesives has steadily increased in recent years. Our previous studies have shown that water-washed cottonseed meal (WCSM) could be used as low-temperature and high-solid content biobased wood adhesives for non-structural bonding as European Standard Class D1 adhesives. In this work, we optimized WCSM-based adhesive formulations with high solid contents up to 49% for bonding of small wood items at low temperatures (40 °C). Chemical denaturing reagents guanidine hydrochloride (GdmCl) and sodium dodecyl sulfate (SDS) were added to improve the bonding capability and viscosity of WCSM. Reviewing industry preferences, four formulations with 20% and 30% of WCSM as well as 9.6% and 19.1% of SDS were used to glue “real world” pencil slat sandwiches for pencil making. All the pencils made from these sandwiches bonded at 40 °C and 1 MPa for 120 min passed the Industrial Temperature Cycle test. The results indicated that WCSM could be used as a low temperature wood adhesive, possibly for the domestic furniture and small utensils niche markets. The pencil sandwich bonding represents a specific application that could benefit from the non-toxic glue and also be an example of an interior application.

Effect of pH and surfactant on the protein: A perspective from theory and experiments

In the present study, we are aimed to explore the bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS) the interaction that can readily show the consequence of the change in concentration of protein with surfactants’ various concentration and the different pH’s, 4.0, 4.7, 7.0 of the medium through the many spectroscopic techniques. The BSA and SDS denaturation fully influenced by the pH. The results interpreted in terms of electrostatic and hydrophobic contributions to the stability of different phases formed in the system. Critical Micelle Concentration (CMC) of surfactant is influenced the protein folding and unfolding. The molecular docking supports the experiment data. This study demonstrates that the above and below the CMC of surfactant can significantly alter the binding interaction with the protein.

2D-LC-MS/MS to measure cleaved high-molecular-weight kininogen in human plasma as a biomarker for C1-INH-HAE.

C1-INH-HAE is caused by activation of plasma kallikrein which subsequently cleaves high-molecular-weight kininogen (HMWK) to generate bradykinin and cHMWK. A novel ion-pair 2D LC-MS/MS assay was developed to measure the 46kDa cHMWK in plasma as a biomarker for C1-INH-HAE. The sample preparation included sodium dodecyl sulfate denaturation, methanol crash, chymotryptic digestion and peptide enrichment by solid phase extraction. The LLOQ was 200ng/ml. The overall cHMWK recovery combining crash and digestion was 57.5%. The precision of the method was ≤12.7% and accuracy ≤-13.8%. A reagent-free LC-MS assay has been developed for the quantitation of 46kDa cHMWK, which was shown to be elevated in plasma of C1-INH-HAE patients due to C1-INH deficiency relative to that of healthy subjects.

Capsule-Targeting Depolymerase, Derived from Klebsiella KP36 Phage, as a Tool for the Development of Anti-Virulent Strategy

The rise of antibiotic-resistant Klebsiella pneumoniae, a leading nosocomial pathogen, prompts the need for alternative therapies. We have identified and characterized a novel depolymerase enzyme encoded by Klebsiella phage KP36 (depoKP36), from the Siphoviridae family. To gain insights into the catalytic and structural features of depoKP36, we have recombinantly produced this protein of 93.4 kDa and showed that it is able to hydrolyze a crude exopolysaccharide of a K. pneumoniae host. Using in vitro and in vivo assays, we found that depoKP36 was also effective against a native capsule of clinical K. pneumoniae strains, representing the K63 type, and significantly inhibited Klebsiella-induced mortality of Galleria mellonella larvae in a time-dependent manner. DepoKP36 did not affect the antibiotic susceptibility of Klebsiella strains. The activity of this enzyme was retained in a broad range of pH values (4.0–7.0) and temperatures (up to 45 °C). Consistently, the circular dichroism (CD) spectroscopy revealed a highly stability with melting transition temperature (Tm) = 65 °C. In contrast to other phage tailspike proteins, this enzyme was susceptible to sodium dodecyl sulfate (SDS) denaturation and proteolytic cleavage. The structural studies in solution showed a trimeric arrangement with a high β-sheet content. Our findings identify depoKP36 as a suitable candidate for the development of new treatments for K. pneumoniae infections.

Online 2D-LC-MS/MS assay to quantify therapeutic protein in human serum in the presence of pre-existing antidrug antibodies.

The formation of antidrug antibodies (ADA) can interfere with the accurate quantitation of therapeutic proteins, leading to significantly underestimated drug concentrations and confounded pharmacokinetic (PK) data interpretation. Although highly desirable, development of ADA-tolerant bioanalytical methods enabling unbiased measurement of both free and ADA-bound drug presents a considerable challenge. We report herein the development and validation of a robust LC-MS assay capable of quantifying therapeutic protein immunoglobulin A1 protease (IgAP) in human serum in the presence of pre-existing anti-IgAP antibodies. The procedure included sodium dodecyl sulfate (SDS) denaturation and chemical reduction of serum proteins to dissociate ADA-drug bindings, followed by tryptic digestion of protein pellets and subsequent LC-MS analysis of the surrogate IgAP peptide using stable isotope labeled peptide internal standard. Substantial enhancements in the sensitivity and selectivity were achieved by the combination of online two-dimensional reversed-phase LC (2D-LC) operated in high and low pH buffers, respectively, for efficient enrichment and quantitation of the surrogate peptide by multiple-reaction monitoring (MRM) mass spectrometry. Unlike ligand-binding assay, our method is not prone to interferences from ADA, allowing accurate and precise measurement of the IgAP in the range of 0.05 to 10 μg/mL in 25 μL of human serum with a wide range of anti-IgAP antibody levels. The intra- and inter-run precision (coefficient of variation (CV%)) was within 11.5% and 10.5%, respectively, and the bias was within ±7.1% for all quality control (QC) concentrations. With little modification, the described method can readily be applicable to the quantitation of other biotherapeutic proteins in the ADA-positive clinical matrices.

Intrinsic Fluorescence Excitation–Emission Matrix Spectral Features of Cottonseed Protein Fractions and the Effects of Denaturants

AbstractTo better understand the functional and physicochemical properties of cottonseed protein, we investigated the intrinsic fluorescence excitation–emission matrix (EEM) spectral features of cottonseed protein isolate (CSPI) and sequentially extracted water (CSPw) and alkali (CSPa) protein fractions, and the effects of denaturants urea, guanidine hydrochloride, and sodium dodecyl sulfate. The EEM showed two contour peaks at the excitation wavelengths of 226 nm (Peak 1) and 277 nm (Peak 2). Addition of denaturants gradually shifted the emission maxima of both peaks from 335 nm to around 353 nm for CSPI and CSPa. The emission maximum (353 nm) of CSPw was unchanged by denaturation. These observations indicated that the tryptophan residues (fluorescence source) in the native CSPI and CSPa were protected within the micro hydrophobic environment, and gradually become water accessible with progressing denaturation. On the other hand, the tryptophan residues in native CSPw were already in contact with water. However, the fluorescence intensity of Peak 1 of all three protein samples decreased with increasing denaturant concentrations, suggesting similarity in some conformational changes in the three samples. Further exploration of the fluorescence mechanism of Peak 1 is needed to understand such similar conformational changes.

Unfolding study of a trimeric membrane protein AcrB

The folding of a multi-domain trimeric α-helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two-state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter-subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate-limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrBΔloop , from the SDS unfolded state. The CD spectrum of the refolded AcrBΔloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild-type AcrB.

Kinetics Study of Curing Epoxy Resins with Hydrolyzed Proteins and the Effect of Denaturants Urea and Sodium Dodecyl Sulfate

The primary goal of this study was to determine the effect of two protein denaturants, urea and sodium dodecyl sulfate (SDS), on the apparent activation energy of cross-linking bisphenol A diglycidyl ether (DGEBA) with hydrolysate of waste animal proteins. Nonisothermal differential scanning calorimetry was used to measure the apparent activation energy of the reactions. The use of SDS resulted in a marked reduction in activation energy, comparable to the reduction in activation energy when a catalyst for epoxy rings, triethylamine (TEA), was used. The addition of urea slightly increased the activation energy. The heat of reaction increased in the presence of SDS because more reactive sites were made available for curing. This work demonstrates the use of SDS as a protein denaturant additive was an energetically efficient alternative to higher degrees of protein hydrolysis for subsequent curing of DGEBA.

Stability of early-stage amyloid-β(1–42) aggregation species

Accumulation of aggregated amyloid-β protein (Aβ) is an important feature of Alzheimer's disease. There is significant interest in understanding the initial steps of Aβ aggregation due to the recent focus on soluble Aβ oligomers. In vitro studies of Aβ aggregation have been aided by the use of conformation-specific antibodies which recognize shape rather than sequence. One of these, OC antiserum, recognizes certain elements of fibrillar Aβ across a broad range of sizes. We have observed the presence of these fibrillar elements at very early stages of Aβ incubation. Using a dot blot assay, OC-reactivity was found in size exclusion chromatography (SEC)-purified Aβ(1–42) monomer fractions immediately after isolation (early-stage). The OC-reactivity was not initially observed in the same fractions for Aβ(1–40) or the aggregation-restricted Aβ(1–42) L34P but was detected within 1–2weeks of incubation. Stability studies demonstrated that early-stage OC-positive Aβ(1–42) aggregates were resistant to 4M urea or guanidine hydrochloride but sensitive to 1% sodium dodecyl sulfate (SDS). Interestingly, the sensitivity to SDS diminished over time upon incubation of the SEC-purified Aβ(1–42) solution at 4°C. Within 6–8days the OC-positive Aβ42 aggregates were resistant to SDS denaturation. The progression to, and development of, SDS resistance for Aβ(1–42) occurred prior to thioflavin T fluorescence. In contrast, Aβ(1–40) aggregates formed after 6days of incubation were sensitive to both urea and SDS. These findings reveal information on some of the earliest events in Aβ aggregation and suggest that it may be possible to target early-stage aggregates before they develop significant stability.

The sclerostin-bone protein interactome

The secreted glycoprotein, sclerostin alters bone formation. To gain insights into the mechanism of action of sclerostin, we examined the interactions of sclerostin with bone proteins using a sclerostin affinity capture technique. Proteins from decalcified rat bone were captured on a sclerostin-maltose binding protein (MBP) amylose column, or on a MBP amylose column. The columns were extensively washed with low ionic strength buffer, and bound proteins were eluted with buffer containing 1M sodium chloride. Eluted proteins were separated by denaturing sodium-dodecyl sulfate gel electrophoresis and were identified by mass spectrometry. Several previously unidentified full-length sclerostin-interacting proteins such as alkaline phosphatase, carbonic anhydrase, gremlin-1, fetuin A, midkine, annexin A1 and A2, and collagen α1, which have established roles in bone formation or resorption processes, were bound to the sclerostin-MBP amylose resin but not to the MBP amylose resin. Other full-length sclerostin-interacting proteins such as casein kinase II and secreted frizzled related protein 4 that modulate Wnt signaling were identified. Several peptides derived from proteins such as Phex, asporin and follistatin that regulate bone metabolism also bound sclerostin. Sclerostin interacts with multiple proteins that alter bone formation and resorption and is likely to function by altering several biologically relevant pathways in bone.

AKAP5 and AKAP12 Form Homo-oligomers.

BackgroundA-kinase-anchoring proteins, AKAPs, constitute a family of scaffolds that play an essential role in catalyzing the spatial-temporal, dynamic interactions of protein kinase A, protein kinase C, tyrosine kinases, G-protein-coupled receptors and ion channels. We studied AKAP5 (AKAP79; MW ~47 kDa) and AKAP12 (gravin, SSECKS; MW ~191 kDa) to probe if these AKAP scaffolds oligomerize.ResultsIn gel analysis and sodium-dodecyl sulfate denaturation, AKAP12 behaved with a MW of a homo-dimer. Only in the presence of the chaotropic agent 8 M urea did gel analysis reveal a monomeric form of AKAP12. By separation by steric-exclusion chromatography, AKAP12 migrates with MW of ~840 kDa, suggestive of higher-order complexes such as a tetramer. Interestingly, the N-(1-840) and C-(840-1782) terminal regions of AKAP12 themselves retained the ability to form dimers, suggesting that the structural basis for the dimerization is not restricted to a single "domain" found within the molecule. In either sodium dodecyl sulfate or urea, AKAP5 displayed a relative mobility of a monomer, but by co-immunoprecipitation in native state was shown to oligomerize. When subjected to steric-exclusion chromatography, AKAP5 forms higher-order complexes with MW ~220 kDa, suggestive of tetrameric assemblies.ConclusionBoth AKAP5 and AKAP12 display the capacity to form supermolecular homo-oligomeric structures that likely influence the localization and function of these molecular scaffolds.

Molecular Basis for the Solvation and Reconstructive Denaturation of Proteins by Detergents

Detergents are widely used for the biochemical and structural study of proteins. Non-ionic and zwitterionic detergents are used as membrane mimetics, where they solvate the hydrophobic regions of integral membrane proteins. In contrast, ionic detergents such as anionic sodium dodecyl sulfate (SDS) and cationic lauryl trimethylammonium chloride (LTAC) are strong protein denaturants that unfold both soluble and membrane proteins. Not only does the SDS-unfolded state have high α-helix content, but SDS also induces α-helix formation irrespective of the intrinsic native secondary structure, a process known as “reconstructive denaturation.” Although this latter phenomenon underpins the ubiquitous technique SDS-PAGE, the mechanism of SDS denaturation and the molecular nature of the SDS denatured state are not known. We use a combined biophysical and computational approach to elucidate the molecular basis of protein denaturation by ionic detergents, with a special focus on the mechanism of reconstructive denaturation by SDS. Specifically, biophysical techniques, including CD and ITC, are used to study the interaction of a set of detergents with model systems that build in complexity from model peptides to full proteins. In the first case, peptide sequence design is used to explore the specific features determining interactions with ionic detergents, while in the latter, chemical modification of residue side chains is used to explore the determinants of detergent interactions in the complex background of a biological protein sequence. In parallel with the biophysical studies, molecular dynamics simulations of the same systems are used to provide atomic resolution detail of the results from the biophysical experiments, and to determine the modes of detergent micelle-protein interaction. Together, these results suggest a consistent mechanism for the reconstructive denaturation phenomenon, for sequence based specificity of ionic detergent interactions, and ultimately, for SDS's universal protein denaturing action.

Ionic strength-dependent denaturation of Thermomyces lanuginosus lipase induced by SDS

Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS–lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.

Characterization of Membrane Protein Non-native States. 1. Extent of Unfolding and Aggregation of Rhodopsin in the Presence of Chemical Denaturants

Little is known about the general folding mechanisms of helical membrane proteins. Unfolded, i.e., non-native states, in particular, have not yet been characterized in detail. Here, we establish conditions under which denatured states of the mammalian membrane protein rhodopsin, a prototypic G protein coupled receptor with primary function in vision, can be studied. We investigated the effects of the chemical denaturants sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride (GuHCl), and trifluoroacetic acid (TFA) on rhodopsin's secondary structure and propensity for aggregation. Ellipticity at 222 nm decreases in the presence of maximum concentrations of denaturants in the order TFA > GuHCl > urea > SDS + urea > SDS. Interpretation of these changes in ellipticity in terms of helix loss is challenged because the addition of some denaturants leads to aggregation. Through a combination of SDS-PAGE, dependence of ellipticity on protein concentration, and 1D (1)H NMR we show that aggregates form in the presence of GuHCl, TFA, and urea but not in any concentration of SDS, added over a range of 0.05%-30%. Mixed denaturant conditions consisting of 3% SDS and 8 M urea, added in this order, also did not result in aggregation. We conclude that SDS is able to prevent the exposure of large hydrophobic regions present in membrane proteins which otherwise leads to aggregation. Thus, 30% SDS and 3% SDS + 8 M urea are the denaturing conditions of choice to study maximally unfolded rhodopsin without aggregation.

Extraction and purification of bovine pancreatic Deoxyribonuclease I

Samples of bovine pancreatic and calf thymus glands were collected from local slaughterers to extract deoxyribonuclease I enzyme (DNase I) and DNA respectively. Crude extract was prepared by using cooled distilled water and 0.25 N sulfuric acid .This study show that the best condition for the crude extract activity was 17 U/ml and 1 hr. Incubated period reaction with its substrate.The bovine pancreatic DNase I was purified by several steps of precipitation using ammonium sulphate to 529.4 times, with an enzymes yield 6.35%. Enzyme characterization studies indicate that: it is active at 2.7 U/ml, and 50 µg /ml DNA after 30 min of reaction time, the enzyme activity was higher at pH 6.5 and it showed stability at pH 9. The maximum enzyme activity was reported at 50 °C The results obtained from the role of metal ions (Mg+2, Mn+2) on enzyme activity indicate that these ions stimulated the enzyme activity while the Ca+2 and Cu+2 had lower stimulating activity on the enzyme but Ag+1 and Hg+2 showed inhibitory effect on enzyme activity. In addition the enzyme activity was inhibited by using denaturing Sodium dodecyl sulphate (SDS), chelating agents Ethylene diamine tetra acetic acid (EDTA), and reducing agents (2-Merceptoethanol and Urea) , and maintained it's activity when incubated with Phenyl Methyl Sulphonyl Floride (PMSF) .

Molecular Basis for the Solvation and Reconstructive Denaturation of Proteins by Detergents

Detergents are widely used for the biochemical and structural study of proteins. Non-ionic and zwitterionic detergents are used as membrane mimetics, where they solvate the hydrophobic regions of integral membrane proteins. In contrast, ionic detergents such as anionic sodium dodecyl sulfate (SDS) and cationic lauryl trimethylammonium chloride (LTAC) are strong protein denaturants that unfold both soluble and membrane proteins. Not only does the SDS-unfolded state have high α-helix content, but SDS also induces α-helix formation irrespective of the intrinsic native secondary structure, a process known as “reconstructive denaturation.” Although this latter phenomenon underpins the ubiquitous technique SDS-PAGE, the mechanism of SDS denaturation and the molecular nature of the SDS denatured state are not known. We use a combined biophysical and computational approach to elucidate the molecular basis of protein denaturation by ionic detergents, with a special focus on the mechanism of reconstructive denaturation by SDS.Specifically, biophysical techniques, including CD and ITC, are used to study the interaction of a set of detergents with model systems that build in complexity from model peptides to full proteins. In the first case, peptide sequence design is used to explore the specific features determining interactions with ionic detergents, while in the latter, chemical modification of residue side chains is used to explore the determinants of detergent interactions in the complex background of a biological protein sequence. In parallel with the biophysical studies, molecular dynamics simulations of the same systems are used to provide atomic resolution detail of the results from the biophysical experiments, and to determine the modes of detergent micelle-protein interaction. Together, these results suggest a consistent mechanism for the reconstructive denaturation phenomenon, for sequence based specificity of ionic detergent interactions, and ultimately, for SDS's universal protein denaturing action.

Chemically Induced Unfolding of Bovine Serum Albumin by Urea and Sodium Dodecyl Sulfate: A Spectral Study with the Polarity‐Sensitive Charge‐Transfer Fluorescent Probe (E)‐3‐(4‐Methylaminophenyl)acrylic Acid Methyl Ester

Sensitivity of the charge-transfer (CT) band of the fluorescence probe (E)-3-(4-methylaminophenyl)acrylic acid methyl ester (MAPAME) towards the polarity of its immediate environment is employed to investigate the binding interaction of the probe with bovine serum albumin (BSA) and uncoiling of BSA by the denaturants urea and sodium dodecyl sulfate micelles. Binding of the probe with BSA produces a blue shift and enhanced intensity of the CT emission band which clearly point toward a decrease in polarity of the immediate environment of MAPAME. This is expected, since binding with BSA moves the probe from a polar water environment to a much less polar, hydrophobic protein interior, where the CT band is expected to be blue-shifted. Higher intensity arises due to fewer non-radiative decay paths available to the probe in the hydrophobic protein environment. Chemically induced unfolding of BSA by urea and sodium dodecyl sulfate is tracked by monitoring the induced spectral changes of the protein-bound probe MAPAME. Red-edge excitation shift or REES, fluorescence resonance energy transfer (FRET) and anisotropy measurements are used to investigate and monitor these binding and unfolding processes.