Reversible Conformational Change Research Articles

Programmable motion of DNA origami mechanisms

DNA origami enables the precise fabrication of nanoscale geometries. We demonstrate an approach to engineer complex and reversible motion of nanoscale DNA origami machine elements. We first design, fabricate, and characterize the mechanical behavior of flexible DNA origami rotational and linear joints that integrate stiff double-stranded DNA components and flexible single-stranded DNA components to constrain motion along a single degree of freedom and demonstrate the ability to tune the flexibility and range of motion. Multiple joints with simple 1D motion were then integrated into higher order mechanisms. One mechanism is a crank-slider that couples rotational and linear motion, and the other is a Bennett linkage that moves between a compacted bundle and an expanded frame configuration with a constrained 3D motion path. Finally, we demonstrate distributed actuation of the linkage using DNA input strands to achieve reversible conformational changes of the entire structure on ∼ minute timescales. Our results demonstrate programmable motion of 2D and 3D DNA origami mechanisms constructed following a macroscopic machine design approach.

Photo-Regulation of Kinesin Intramolecularly Crosslinked by Bifunctional Azobenzne Derivative at the Coiled-Coil Stalk Region

Kinesin is an ATP driven dimeric motor protein carries cellular cargoes along microtubules. The stalk region of kinesin is responsible for dimerization with coiled-coil interaction. Formation of dimer is essential for kinesin to perform processive movement along the microtubules. Aim of this study is to control dimerization of kinesin by the reversible conformational change at the coild-coil stalk region using photochromic molecule resulting in photo-reversible regulation of motility. Azobenzene-dimaleimide (ABDM) is a bifunctional SH reactive photochromic crosslinker and its crosslinking span is altered by cis-trans photo-isomerization of azobenznene moiety upon ultraviolet and visible light irradiations. We have previously demonstrated that the two reactive cysteine residues SH1(707) and SH2(697) in α-helix of myosin which region is believed to have a energy transducing role, were cross-linked by ABDM. And cis-trans isomerization of the crosslinked ABDM induced disordered α-helix in the SH1-SH2 region resulting in global conformational change of myosin head.In this study, we prepared kinesin mutant K380 cys light which has truncated minimum stalk to form dimer. Subsequently, Lys360 and Ala361 in the coiled-coli region of the mutant K380 were substituted by cysteine. The kinesin mutant K380(K350C,A361C) was modified with ABDM. The intermolecular and intramolecular crosslinking were analyzed SDS-PAGE. The optimal condition to crosslinking was 10 times excess molar ABDM for kinesin mutant and modification for 160 min at room temp. The conformational change of the ABDM crosslinked kinesin induced photoisomerization was studied with CD spectroscopy. And the photo-reversible dimerization of ABDM crosslinked kinesin was examined.

Some methodical aspects in investigations on wheat gluten

3 M urea has been shown to cause considerable, and only partially reversible conformational changes of gluten molecules. Homogenization has proved to act mechanically, breaking down some molecular bonds. No structural changes could be observed during freeze drying gluten, as well as after brief heating of its acetic acid extracts.

Probing Temperature Dependent Conformation Change of Calmodulin Protein using Molecular Dynamics Simulations

With advancement in computing hardware and simulation techniques scientists have developed a substantial overlap between the timescales accessible to atomic-level simulations and those on which the fastest-biological actions take place. In this work we probe the temperature dependent behavior of Calmodulin (CaM) protein using Molecular dynamics (MD). CaM plays an important role in calcium signaling pathways in the eukaryotic cells as it can bind to a variety of other proteins or small organic compounds. Environmental conditions such as temperature would play an important role in the dynamics of this protein. Earlier such kinds of studies have been performed experimentally using fluorescence measurements as in. The calcium bound form of the protein CaM is observed to undergo a reversible conformation change in the temperature range 22-28°C. Leap-dynamics approach was used earlier to study the temperature dependent conformation change of CaM. In this work we perform extensive MD simulations of 100 ns each at various temperatures ranging 280-360 K on the 3CLN and 1CFD forms of calmodulin. A remarkable dependence of folding and unfolding on the temperature and initial structure is obtained similar to as reported in our earlier study. The linker is observed to be more flexible in the case of 1CFD leading to a bend compact form during the simulation at 325 K. 3CLN on the other hand tries to remain in a state close to its initial extended structure. Normal mode analysis is also performed to understand the type of motion experienced by the protein during the MD simulation.

Dynamic Visualization of α-Catenin Reveals Rapid, Reversible Conformation Switching between Tension States

The cytosolic protein α-catenin is a postulated force transducer at cadherin complexes. The demonstration of force activation, identification of consequent downstream events in live cells, and development of tools to study these dynamic processes in living cells are central to elucidating the role of α-catenin in cellular mechanics and tissue function. Here we demonstrate that α-catenin is a force-activatable mechanotransducer at cell-cell junctions by using an engineered α-catenin conformation sensor based on fluorescence resonance energy transfer (FRET). This sensor reconstitutes α-catenin-dependent functions in α-catenin-depleted cells and recapitulates the behavior of the endogenous protein. Dynamic imaging of cells expressing the sensor demonstrated that α-catenin undergoes immediate, reversible conformation switching in direct response to different mechanical perturbations of cadherin adhesions. Combined magnetic twisting cytometry with dynamic FRET imaging revealed rapid, local conformation switching upon the mechanical stimulation of specific cadherin bonds. At acutely stretched cell-cell junctions, the immediate, reversible conformation change further reveals that α-catenin behaves like an elastic spring in series with cadherin and actin. The force-dependent recruitment of vinculin—a principal α-catenin effector—to junctions requires the vinculin binding site of the α-catenin sensor. In cells, the relative rates of force-dependent α-catenin conformation switching and vinculin recruitment reveal that α-catenin activation and vinculin recruitment occur sequentially, rather than in a concerted process, with vinculin accumulation being significantly slower. This engineered α-catenin sensor revealed that α-catenin is a reversible, stretch-activatable sensor that mechanically links cadherin complexes and actin and is an indispensable player in cadherin-specific mechanotransduction at intercellular junctions.

Structural Mechanism of Nuclear Transport Mediated by Importin β and Flexible Amphiphilic Proteins

Karyopherin β family proteins mediate the nuclear/cytoplasmic transport of various proteins through the nuclear pore complex (NPC), although they are substantially larger than the size limit of the NPC.To elucidate the molecular mechanism underlying this paradoxical function, we focused on the unique structures called HEAT repeats, which consist of repetitive amphiphilic α helices. An in vitro transport assay and FRAP analyses demonstrated that not only karyopherin β family proteins but also other proteins with HEAT repeats could pass through the NPC by themselves, and serve as transport mediators for their binding partners. Biochemical and spectroscopic analyses and molecular dynamics simulations of purified HEAT-rich proteins revealed that they interact with hydrophobic groups, including phenyl and alkyl groups, and undergo reversible conformational changes in tertiary structures, but not in secondary structures. These results show that conformational changes in the flexible amphiphilic motifs play a critical role in translocation through the NPC.

Facile one-pot preparation of gold nanoparticles in the presence of fluoroalkyl end-capped oligomers, fluoroalkyl end-capped oligomers/silica nanocomposites, and fluoroalkyl end-capped oligomers/polyaniline nanocomposites

A variety of fluoroalkyl end-capped oligomers, such as fluoroalkyl end-capped acrylic acid oligomer [RF-(ACA) n -RF], acryloylmorpholine oligomer [RF-(ACMO) n -RF], 2-acrylamido-2-methylpropanesulfonic acid oligomer [RF-(AMPS) n -RF], 2-(methacryloyloxy)ethanesulfonic acid oligomer [RF-(MES) n -RF], and N,N-dimethylacrylamide oligomer [RF-(DMAA) n -RF], were applied to the autoreduction of gold ions to give the corresponding oligomers/gold nanocomposites, of whose sharp plasmon absorption bands are observed around 535 nm. In these fluorinated oligomers, RF-(ACA) n -RF oligomer and RF-(ACMO) n -RF were effective for the one-pot preparation of the gold nanoparticles under very mild conditions; although the other fluorinated oligomers and the corresponding non-fluorinated–(ACMO) n -oligomer were unable to afford the gold nanoparticles. RF-(ACA) n -RF/SiO2 nanocomposites and RF-(ACMO) n -RF/SiO2 nanocomposites, which were prepared by the sol–gel reactions of tetraethoxysilane in the presence of silica nanoparticles and the corresponding oligomers under alkaline conditions, were also applied to the encapsulation of gold nanoparticles into these fluorinated nanocomposite cores through the autoreduction of gold ions at room temperature. Interestingly, these fluorinated oligomers/silica nanocomposite-encapsulated gold nanocomposites before and after calcination at 800 °C were found to exhibit the same plasmon absorption band around 525 nm. RF-(MES) n -RF oligomer and RF-(AMPS) n -RF oligomer are not suitable for the autoreduction of gold ions; however, RF-(MES)n-RF[or RF-(AMPS) n -RF]/polyaniline [PAn] nanocomposites, which were prepared by the polymerization of aniline initiated by ammonium persulfate in the presence of the corresponding oligomer, enabled the formation of gold nanoparticles through the oxidation of PAn in the composites at room temperature. The reversible conformational change of PAn in the nanocomposites from the polyemeraldine salt to the oxidized pernigraniline base was observed during such oxidation process.

Topography-driven bionano-interactions on colloidal silica nanoparticles

We report here that the surface topography of colloidal mesoporous silica nanoparticles (MSNs) plays a key role on their bionano-interactions by driving the adsorption of biomolecules on the nanoparticle through a matching mechanism between the surface cavities characteristics and the biomolecules stereochemistry. This conclusion was drawn by analyzing the biophysicochemical properties of colloidal MSNs in the presence of single biomolecules, such as alginate or bovine serum albumin (BSA), as well as dispersed in a complex biofluid, such as human blood plasma. When dispersed in phosphate buffered saline media containing alginate or BSA, monodisperse spherical MSNs interact with linear biopolymers such as alginate and with a globular protein such as bovine serum albumin (BSA) independently of the surface charge sign (i.e. positive or negative), thus leading to a decrease in the surface energy and to the colloidal stabilization of these nanoparticles. In contrast, silica nanoparticles with irregular surface topographies are not colloidally stabilized in the presence of alginate but they are electrosterically stabilized by BSA through a sorption mechanism that implies reversible conformation changes of the protein, as evidenced by circular dichroism (CD). The match between the biomolecule size and stereochemistry with the nanoparticle surface cavities characteristics reflects on the nanoparticle surface area that is accessible for each biomolecule to interact and stabilize any non-rigid nanoparticles. On the other hand, in contact with variety of biomolecules such as those present in blood plasma (55%), MSNs are colloidally stabilized regardless of the topography and surface charge, although the identity of the protein corona responsible for this stabilization is influenced by the surface topography and surface charge. Therefore, the biofluid in which nanoparticles are introduced plays an important role on their physicochemical behavior synergistically with their inherent characteristics (e.g., surface topography).

Dual-responsive polymer brushes as an electrochemical logic gate

Stimuli-responsive polymer brushes modified surfaces and binary polymer surfaces can be created on an initiator terminated substrate via surface initiated atom-transfer radical polymerization (SI-ATRP), in which the growth kinetics can be modulated by Cu(II) ratio. As stimuli-responsive brushes, many environmental triggers can drive their conformation change such as pH and salt concentration. The external-stimuli modulated reversible conformational changes of polymer brushes can influence the charge transport behavior investigated by electrochemistry method, which have potential applications in in situ switching ion permeability and charge transport. The charge transport of binary polymer brush can be switched on or off by different ion flexibly which is an electrochemical logic switch.

Dynamic contact angle analysis of protein adsorption on polysaccharide multilayer's films for biomaterial reendothelialization.

Atherosclerosis is a major cardiovascular disease. One of the side effects is restenosis. The aim of this work was to study the coating of stents by dextran derivates based polyelectrolyte's multilayer (PEM) films in order to increase endothelialization of injured arterial wall after stent implantation. Films were composed with diethylaminoethyl dextran (DEAE) as polycation and dextran sulphate (DS) as polyanion. One film was composed with 4 bilayers of (DEAE-DS)4 and was labeled D−. The other film was the same as D− but with an added terminal layer of DEAE polycation: (DEAE-DS)4-DEAE (labeled D+). The dynamic adsorption/desorption of proteins on the films were characterized by dynamic contact angle (DCA) and atomic force microscopy (AFM). Human endothelial cell (HUVEC) adhesion and proliferation were quantified and correlated to protein adsorption analyzed by DCA for fibronectin, vitronectin, and bovine serum albumin (BSA). Our results showed that the endothelial cell response was optimal for films composed of DS as external layer. Fibronectin was found to be the only protein to exhibit a reversible change in conformation after desorption test. This behavior was only observed for (DEAE-DS)4 films. (DEAE-DS)4 films could enhance HUVEC proliferation in agreement with fibronectin ability to easily change from conformation.

Switching the Antimicrobial Activity of Gramicidin S by Light

Light-driven reversible conformational changes of proteins are of fundamental importance to nature. Here, we present the development of a photo-controllable antimicrobial peptide (AMP), based on a novel photo-switch in the backbone. AMPs are an essential part of innate immune system of living organisms and defend them against various bacteria, viruses, and fungi. They are promising therapeutic sources for future antibiotics as they permeabilize bacterial membranes, but they tend to suffer from cytotoxic side effects against eukaryotic cells. These problems, preventing clinical applications, could be eliminated by remotely switching the membranolytic activity ON and OFF. We have thus designed and synthesized a reversibly photo-isomerizable amino acid analogue based on a diarylethylene scaffold. The amino acid analogue was incorporated into the cyclic backbone of the antimicrobial peptide Gramicidin S. Several peptidomimetics were successfully synthesized, their photochromic features were recorded in the ON and OFF states, and the corresponding molecular conformations were analyzed by CD spectroscopy and MD simulations. Antimicrobial assays and hemolysis tests showed that the biological activity could be directly controlled by irradiation with visible and UV light. These results open the way to develop new strategies for treating bacterial infections or other localized pathologies, by activating the antibiotic with light only in those specific areas where needed. The use of such ‘smart’ antibiotics will largely eliminate the harmful side effects on the whole body and should also prevent the concomitant development of bacterial resistance.

Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5′-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic alpha helices, including a short helix 3′. The ‘helix–short helix–helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly upregulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.

Azobenzamide-based proteomorphous objects as a light/pH-induced photoswitchable module

Azobenzamide-based molecular scaffolds viz. (E)-4,4′(diazene-1,2-diyl)bis(N-pyridin-3-yl)benzamide (1) and (E)-4,4′(diazene-1,2-diyl)bis(N-pyridin-4-yl)benzamide (2), yield proteomorphous architectures i.e., secondary “β-pleated sheet” and tertiary “β-hairpin” respectively, that offer “optical data storage” competence as a result of reversible light-induced conformational changes in organic solvent.

Helical poly(phenylacetylene)s containing schiff-base and amino groups: Synthesis, secondary structures, and responsiveness to benzoic acid

Novel acetylenic monomers containing Schiff-base and amino groups, (S)-N-(4-ethynylbenzylidene)-1-phenylethanamine (1a), (R)-N-(4-ethynylbenzylidene)-1-phenylethanamine (1b), N-(4-ethynylbenzylidene)-1-phenylethanamine (1c), (R)-N-(4-ethynylbenzyl)-1-phenylethanamine (1d), and (R)-N-(4-ethynylbenzyl)-1-phenylethanamine (1e) were synthesized and polymerized with [(nbd)RhCl]2/Et3N catalyst to afford the corresponding polymers 2a-e with moderate molecular weights (Mn = 9000–60,000) in high yields (85–97%). All the polymers were soluble in common organic solvents including toluene, CHCl3, CH2Cl2, THF, and DMF. Large optical rotations and strong CD signals demonstrated that 2a, 2b, 2d, and 2e take helical structures with a predominantly one-handed screw sense. The effects of solvents and temperature revealed that these polymers took dynamic helical structure based on the steric effect of side groups. The CD patterns of 2d and 2e containing free amino moieties were completely inverted by the addition of benzoic acid. Upon further addition of NaOH, the CD pattern returned to the original one, indicating the reversible conformational change of these polymers according to pH. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 5248–5256

Nearly Reversible Conformational Change of Amyloid Fibrils as Revealed by pH-Jump Experiments

pH-jump induced conformational transitions between substates of preformed amyloid fibrils made by a fragmented peptide of helix 2 (H2 peptide) of MoPrP were detected, and their kinetics were analyzed using a novel pH-jump apparatus specially designed for observing amyloids. Previously, we reported that H2 peptide formed ordered fibrils with a minimum at 207 nm on CD spectra at pH 2.9 (named pH 2.9 fibrils), but formed aggregate-like fibrils with a minimum at 220 nm at pH 7.5 (named pH 7.5 fibrils). When pH-jump from 2.9 to 7.5 was performed, the CD spectrum changed instantly, but the finally observed ellipticities were clearly distinct from those of pH 7.5 fibrils. Thus, the finally observed state is termed 'pH 7.5-like fibrils'. However, pH 7.5-like fibrils reverted to the conformation very similar to that of the pH 2.9 fibrils when the pH of the solution was restored to 2.9. Then, we examined the kinetics of the nearly reversible conformational changes between pH 2.9 fibrils and pH 7.5-like fibrils using ANS fluorescence stopped-flow, and we observed relatively fast phases (0.7-18 s(-1)). In contrast, the conversion between pH 7.5-like fibrils and pH 7.5 fibrils never occurred (<0.2 day(-1)). Thus, H2 fibrils can be switched readily between distinct conformations separated by a low energy barrier, while a large energy barrier clearly separated the different conformations. These conformational varieties of amyloid fibrils may explain the physical basis of the diversity in prion.

TBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation

Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death.

STIMULI RESPONSIVE MORPHOLOGICAL CHANGES OF PNIPA POLYMER BRUSHES SYNTHESIZED ON SILICON SUBSTRATE

High-density polymer brushes were grown from the silicon surface by atom transfer radical polymerization of Poly(N-isopropylacrylamide) (PNIPA) at different polymerization conditions. PNIPA brushes were prepared using Copper (I) Chloride/tris(2-(dimetylamino)ethyl)amine (Me6TREN) as a catalytic system in DMSO at 20°C. Free polymer formed during the brush formation was characterized by gel permeation chromatography. The grafting densities up to 0.52 chains/nm2 were obtained. The layer thickness of polymer brush increases with the increase of conversion of the monomer conversion as well as polymerization time. Atomic force microscopy and air bubble contact angle under pH solution were employed to study the surface morphology, reversible conformational changes of and stimulus-response behavior. PNIPA brushes exhibited a different nanomorphology after treatment with different pH solution. It also revealed a unique reversible wetting behavior with pH. The reversible properties of the PNIPA brushes can be used to regulate the adsorption of the sulfonated PS nanoparticles.

Synthesis and Photoresponse of Helically Folded Poly(phenyleneethynylene)s Bearing Azobenzene Moieties in the Main Chains

Novel optically active poly(phenyleneethynylene)s bearing azobenzene moieties in the main chains [poly(1–2m), poly(1–2p)] were synthesized by the Sonogashira–Hagihara coupling polymerization of 3′,5′-diiodo-4′-hydroxy-N-α-tert-butoxycarbonyl-d-phenylglycine hexylamide (1) with 3,3′-diethynylazobenzene (2m) and 4,4′-diethynylazobenzene (2p). The corresponding polymers [poly(1–2m), poly(1–2p)], with number-average molecular weights of 10700 and 9400, were obtained in 70% and 86% yields, respectively. CD and UV–vis spectroscopic analyses revealed that poly(1–2m) and poly(1–2p) formed predominantly one-handed helically folded structures in CHCl3/THF mixtures. Poly(1–2m) underwent a reversible conformational change between folded and unfolded structures upon UV and visible irradiation, as a result of trans–cis isomerization of the azobenzene moieties. On the other hand, poly(1–2p) showed very little conformational transformation or azobenzene isomerization. The formation of helical structures was supported by conformational analysis based on the molecular mechanics (MM), semiempirical molecular orbital (MO), and density functional theory (DFT) methods.

Synthesis of copolymer based on naphthalene diimide connected with a non-conjugated flexible linker

We synthesized a copolymer in which segments of naphthalene diimide derivatives are connected by a short flexible linker that allow neighboring segments to interact closely with each other. Comparison of the optoelectronic properties of the copolymer and the monomer indicates weak intramolecular π–π interaction between NDI segments in the copolymer. Variable-temperature UV–vis absorption spectra measured in solution suggest a reversible change in the intramolecular interaction of the NDI segments in the polymers, implying a reversible conformation change.

Chaperones Rejuvenate Folding and Activity of 3-β Hydroxysteroid Dehydrogenase 2

The steroidogenic enzyme 3-β hydroxysteroid dehydrogenase 2 (3βHSD2) mediates the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione through both its dehydrogenase and isomerase activities, making it necessary for the protein to undergo a reversible conformational change. We hypothesized that chaperones assist 3βHSD2 in switching between the conformations to initiate, enhance, and maintain activity. In the presence of the chaperone lauryl maltoside (LM), 3βHSD2 immediately converted pregnenolone to progesterone, with a 6.4-fold increase in synthesis. Using far-UV circular dichroism (CD), we found that addition of LM increased 3βHSD2's α-helical content, which over time reverted to control levels, suggesting the formation of a stable but reversible conformation possibly due to hydrophobic interactions of the protein with LM micelles. We also found that LM increased fluorescence resonance energy transfer (FRET) about 11-fold between 3βHSD2 and fluorescing ANS molecules. This observation supports the idea that detergent(s) act as chaperones to assist 3βHSD2 in forming stable complexes, which in turn promotes proper folding. Mass spectrometric fingerprinting illustrated that LM incubation resulted in an ordered fragmentation of molecular mass from 39 to 13 kDa, as compared to limited or no proteolysis in the absence of LM. In addition, space-filling modeling demonstrated that 3βHSD2 association with detergents likely exposed the hydrophobic region, leading to its proteolysis. We conclude that detergents help 3βHSD2 to refold in order to rejuvenate, contributing to the ability of cells to rapidly produce steroids when needed.