Native-like Structure Research Articles

Refolding dynamics and immunoinformatic insights into Vibrio cholerae OmpA, OmpK, and OmpV for vaccine applications.

OmpA, OmpK, and OmpV are crucial for the pathogenesis of Vibrio cholerae, functioning within the bacterium's outer membrane; they present significant potential as candidates for vaccine development. Due to their intrinsic β-sheet richness, these OMPs tend to form inclusion bodies whenever overexpression is attempted. To achieve a native-like structure, detergents can be utilized during the refolding of OMPs from inclusion bodies. The impact of different detergents is examined on the renaturation of these OMPs, specifically non-ionic and zwitterionic detergents. The findings provide valuable insights into detergent selection, with LDAO and DDM emerging as the best protein refolding agents, facilitating successful structural and functional studies of these OMPs. Furthermore, using immunoinformatics it is established that OmpA, OmpK, and OmpV carry B- and T-cell epitopes in their exposed extracellular regions. The presence of immunodominant regions makes it easier to employ these proteins as vaccine candidates as they are stable, non-allergenic, and likely to stimulate successful innate and active immune responses. Overall, with all three OMPs harboring numerous immunogenic epitopes, they can be employed in subunit vaccines against Vibrio spp. and contribute to the development of diagnostic tools for effective disease mitigation.

CT584 Is Not a Protective Vaccine Antigen against Respiratory Chlamydial Challenge in Mice.

Background:Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen in humans worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and decentralized production of recombinant protein vaccine antigens. Methods: Here, we use CFPS to produce the full-length putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for evaluation as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) (RIBM, Tsukuba, Japan) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Results: Immunization with CT584 generated robust antibody responses but weak cell-mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lung weights, and the presence of high numbers of IFUs in the lungs. Conclusion: While CT584 was not a protective vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens make it an attractive technique for antigen production.

Molecular mechanism of alpha- and beta-cyclodextrin assisted refolding of SDS-induced amyloid fibrils of myoglobin

The formation of amyloids arises from the misfolding or unfolding of various proteins. This study investigated the impact of chemical chaperones, alpha-cyclodextrin (α-CD) and beta-cyclodextrin (β-CD), on the solubilization and defibrillation of sodium dodecyl sulfate (SDS)-induced myoglobin (Mb) amyloid fibrils. Amyloid fibril was developed in Mb in the presence of 0.8 mmol dm−3 SDS at pH 4.5. Subsequently, SDS-induced Mb amyloid was incubated with varying concentrations of α-CD and β-CD at the same pH. Our turbidity, intrinsic fluorescence, SDS-PAGE, and transmission electron microscope data showed that α-CD (2.1 mmol dm−3) was more efficiently solubilized the SDS-treated Mb aggregates than β-CD (4.0 mmol dm−3). But, another interesting phenomenon was observed with β-CD. The ThT fluorescence and far-UV CD data suggested that amyloid defibrillation by β-CD (0.6 mmol dm−3) was more effective than α-CD (0.9 mmol dm−3). The intrinsic fluorescence and far-UV CD data also showed that SDS-treated Mb regained native-like structure at lower α-CD compared to β-CD. Overall our data suggest that α-CD was more efficient in aggregate solubilization while β-CD was more effective in amyloid defibrillation. Understanding the mechanisms underlying cyclodextrins’ actions may serve as a basis for identifying potential drugs to suppress amyloid fibril formation effectively. This research provides valuable insights into mitigating amyloid-related pathologies.

Bioprinting of anisotropic functional corneal stroma using mechanically robust multi-material bioink based on decellularized cornea matrix

Corneal scarring is a common cause of blindness, affecting millions globally each year. A huge gap between the demand and supply of donor tissue currently limits corneal transplantation, the only definitive therapy for patients with corneal scarring. To overcome this challenge, researchers have harnessed the efficacy of 3D bioprinting to fabricate artificial corneal stromal constructs. With all the different bioinks available, the decellularized corneal matrix-based bioprinted construct can fulfill the required biological functionality but is limited by the lack of mechanical stiffness. Additionally, from a biophysical standpoint, it is necessary for an ideal corneal substitute to mimic the anisotropy of the cornea from the central optic zone to the surrounding periphery. In this study, we enhanced the mechanical robustness of decellularized cornea matrix (DCM) hydrogel by blending it with another natural polymer, sonicated silk fibroin solution in a defined ratio. Although hybrid hydrogel has an increased complex modulus than DCM hydrogel, it has a lower in vitro degradation rate and increased opaqueness due to the presence of crystalline beta-sheet conformation within the hydrogel. Therefore, we used this multi-material bioink-based approach to fabricate a corneal stromal equivalent where the outer peripheral corneal rim was printed with a mechanically robust polymeric blend of DCM and sonicated silk fibroin and the central optic zone was printed with only DCM. The bioprinted corneal stroma thus maintained its structural integrity and did not break when lifted with forceps. The two different bioinks were encapsulated with human limbus-derived mesenchymal stem cells (hLMSC) individually and 3D bioprinted in different patterns (concentric and parallel) to attain a native-like structure in terms of architecture and transparency. Thus, the bilayer cornea constructs maintained high cell viability and expressed keratocyte core proteins indicating optimal functionality. This approach helped to gain insight into bioprinting corneas with heterogeneous mechanical property without disturbing the structural clarity of the central optic zone.

Revealing the fates of proteins in the gas phase

The ability to observe intact proteins by native mass spectrometry allows measurements of size, oligomeric state, numbers and types of ligands and post translational modifications bound, among many other characteristics. These studies have the potential to, and in some cases are, advancing our understanding of the role of structure in protein biology and biochemistry. However, there are some long-unresolved questions about to what extent solution-like structures persist without solvent in the vacuum of the mass spectrometer. Strong evidence from multiple sources over the years has demonstrated that well-folded proteins maintain native-like states if care is taken during sample preparation, ionization, and transmission through the gas phase. For partially unfolded states, dynamic and disordered proteins, and other important landmarks along the protein folding/unfolding pathway, caution has been urged in the interpretation of the results of native ion mobility/mass spectrometric data. New gas-phase tools allow us to provide insight into these questions with in situ, in vacuo labeling reactions delivered through ion/ion chemistry. This Young Scientist Perspective demonstrates the robustness of these tools in describing native-like structure as well as possible deviations from native-like structure during native ion mobility/mass spectrometry. This Perspective illustrates some of the changes in structure produced by the removal of solvent and details some of the challenges and potential of the field.

The Curious Case of A31P, a Topology-Switching Mutant of the Repressor of Primer Protein: A Molecular Dynamics Study of Its Folding and Misfolding.

The effect of mutations on protein structures is usually rather localized and minor. Finding a mutation that can single-handedly change the fold and/or topology of a protein structure is a rare exception. The A31P mutant of the homodimeric Repressor of primer (Rop) protein is one such exception: This single mutation ─and as demonstrated by two independent crystal structure determinations─ can convert the canonical (left-handed/all-antiparallel) 4-α-helical bundle of Rop to a new form (right-handed/mixed parallel and antiparallel bundle) displaying a previously unobserved "bisecting U" topology. The main problem with understanding the dramatic effect of this mutation on the folding of Rop is to understand its very existence: Most computational methods appear to agree that the mutation should have had no appreciable effect, with the majority of energy minimization methods and protein structure prediction protocols indicating that this mutation is fully consistent with the native Rop structure, requiring only a local and minor change at the mutation site. Here we use two long (10 μs each) molecular dynamics simulations to compare the stability and dynamics of the native Rop versus a hypothetical structure that is identical with the native Rop but is carrying this single Alanine31 to Proline mutation. Comparative analysis of the two trajectories convincingly shows that, in contrast to the indications from energy minimization ─but in agreement with the experimental data─, this hypothetical native-like A31P structure is unstable, with its turn regions almost completely unfolding, even under the relatively mild 320 K NpT simulations that we have used for this study. We discuss the implication of these findings for the folding of the A31P mutant, especially with respect to the proposed model of a double-funneled energy landscape.

Evaluation in mice of cell-free produced CT584 as a Chlamydia vaccine antigen.

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and de-centralized production of recombinant protein vaccine antigens. Here, we use CFPS to produce the putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for use as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four-weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Immunization with CT584 generated robust antibody responses but weak cell mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lungs' weights and the presence of high numbers of IFUs in the lungs. While CT584 alone may not be the ideal vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens makes it an attractive technique for antigen production.

Exploring the Specific Role of Iron Center in the Catalytic Activity of Human Serum Transferrin: CTAB-Induced Conformational Changes and Sequestration by Mixed Micelles.

Conformational changes play a seminal role in modulating the activity of proteins. This concept becomes all the more relevant in the context of metalloproteins, owing to the formation of specific conformation(s) induced by internal perturbations (like a change in pH, ligand binding, or receptor binding), which may carry out the binding and release of the metal ion/ions from the metal binding center of the protein. Herein, we investigated the conformational changes of an iron-binding protein, monoferric human serum transferrin (Fe-hTF), using several spectroscopic approaches. We could reversibly tune the cetyltrimethylammonium bromide (CTAB)-induced conformation of the protein, exploiting the concept of mixed micelles formed by three sequestrating agents: (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) hydrate (CHAPS) and two bile salts, namely, sodium cholate (NaC) and sodium deoxycholate (NaDC). The formation of mixed micelles between CTAB and these reagents (CHAPS/NaC/NaDC) results in the sequestration of CTAB molecules from the protein environment and aids the protein in reattaining its native-like structure. However, the guanidinium hydrochloride-induced denatured Fe-hTF did not acquire its native-like structure using these sequestrating agents, which substantiates the exclusive role of mixed micelles in the present study. Apart from this, we found that the conformation of transferrin (adopted in the presence of CTAB) displays pronounced esterase-like activity toward the para-nitrophenyl acetate (PNPA) substrate as compared to native transferrin. We also outlined the impact of the iron center and amino acids surrounding the iron center on the effective catalytic activity in the CTAB medium. We estimated ∼3 times higher specific catalytic efficiency for the iron-depleted Apo-hTF compared to the fully iron-saturated Fe2-hTF in the presence of CTAB.

Methotrexate for Drug Repurposing as an Anti-Aggregatory Agent to Mercuric Treated α-Chymotrypsinogen-A.

Protein aggregation is related to numerous pathological conditions like Alzheimer's and Parkinson's disease. In our study, we have shown that an already existing FDA-approved drug; methotrexate (MTX) can be reprofiled on preformed α-chymotrypsinogen A (α-Cgn A) aggregates. The zymogen showed formation of aggregates upon interaction with mercuric ions, with increasing concentration of Hg2Cl2 (0-150 µM). The hike in ThT and ANS fluorescence concomitant with blue shift, bathochromic shift and the hyperchromic effect in the CR absorbance, RLS and turbidity measurements, substantiate the zymogen β-rich aggregate formation. The secondary structural alterations of α- Cgn A as analyzed by CD measurements, FTIR and Raman spectra showed the transformation of native β-barrel conformation to β-inter-molecular rich aggregates. The native α- Cgn A have about 30% α-helical content which was found to be about 3% in presence of mercuric ions suggesting the formation of aggregates. The amorphous aggregates were visualized by SEM. On incubation of Hg2Cl2 treated α- Cgn A with increasing concentration of the MTX resulted in reversing aggregates to the native-like structure. These results were supported by remarkable decrease in ThT and ANS fluorescence intensities and CR absorbance and also consistent with CD, FTIR, and Raman spectroscopy data. MTX was found to increase the α-helical content of the zymogen from 3 to 15% proposing that drug is efficient in disrupting the β-inter-molecular rich aggregates and reverting it to native like structure. The SEM images are in accordance with CD data showing the disintegration of aggregates. The most effective concentration of the drug was found to be 120 µM. Molecular docking analysis showed that MTX molecule was surrounded by the hydrophobic residues including Phe39, His40, Arg145, Tyr146, Thr151, Gly193, Ser195, and Gly216 and conventional hydrogen bonds, including Gln73 (bond length: 2.67Å), Gly142 (2.59Å), Thr144 (2.81Å), Asn150 (2.73Å), Asp153 (2.71Å), and Cys191 (2.53Å). This investigation will help to find the use of already existing drugs to cure protein misfolding-related abnormalities.

Strontium-loaded 3D intramedullary nail titanium implant for critical-sized femoral defect in rabbits.

The treatment of critical-sized bone defects has long been a major problem for surgeons. In this study, an intramedullary nail shaped three-dimensional (3D)-printed porous titanium implant that is capable of releasing strontium ions was developed through a simple and cost-effective surface modification technique. The feasibility of this implant as a stand-alone solution was evaluated using a rabbit's segmental diaphyseal as a defect model. The strontium-loaded implant exhibited a favorable environment for cell adhesion, and mechanical properties that were commensurate with those of a rabbit's cortical bone. Radiographic, biomechanical, and histological analyses revealed a significantly higher amount of bone ingrowth and superior bone-bonding strength in the strontium-loaded implant when compared to an untreated porous titanium implant. Furthermore, one-year histological observations revealed that the strontium-loaded implant preserved the native-like diaphyseal bone structure without failure. These findings suggest that strontium-releasing 3D-printed titanium implants have the clinical potential to induce the early and efficient repair of critical-sized, load-bearing bone defects.

Directing ligament-mimetic bi-directional cell organization in scaffolds through zone-specific microarchitecture for ligament tissue engineering

Ligament tissues exhibit zone-specific anisotropic cell organization. The cells in ligament-proper are longitudinally oriented, whereas, the cells in epiligament are circumferentially oriented. Therefore, scaffolds developed to regenerate ligament tissues should possess adequate architectural features to govern ligament-mimetic bi-directional cell organization. The scaffold architectural features along with ligament-mimetic cell organization may ultimately yield neo-tissues with ligament-like extracellular matrix (ECM) structure and biomechanical properties. Towards this goal, we fabricated a silk/gelatin-based core–shell scaffold (csSG) with zone-specific anisotropic architectural features, wherein, the core of the scaffold possessed longitudinally aligned pores while the shell of the scaffold possessed parallel microgrooves that are aligned circumferentially around the surface of the scaffold. The ligament-mimetic architectural features significantly improved the mechanical properties of the scaffold. Moreover, architectural features of the csSG scaffold governed zone-specific anisotropic organization of cells. The cells in the core were longitudinally oriented as observed in the ligament-proper and the cells on the shell were circumferentially oriented as observed in epiligament. This bi-directional cell orientation partially mimicked the complex cellular network in native ligament tissue. Additionally, both the core and the shell individually supported fibrogenic differentiation of stem cells which further improved their potential for ligament tissue engineering. Further, the aligned pores of the core could govern unidirectional organization of ECM deposited by cells which is crucial for regenerating anisotropic tissues like ligaments. Finally, when implanted subcutaneously in mice, the scaffolds retained their anisotropic architecture for at least 2 weeks, were biocompatible, supported cell infiltration and governed anisotropic organization of cells and ECM. Taken together, the fabricated biomimetic csSG scaffold, through its zone-specific architectural features, could govern ligament-mimetic cellular and ECM organization which is ultimately expected to achieve regeneration of ligament tissues with native-like hierarchical structure and biomechanical properties. Consequently, this study introduces bi-directional structural parameters as design criteria for developing scaffolds for ligament tissue engineering.

Determining native-like membrane protein structures from cell membrane-derived vesicles

Determining native-like membrane protein structures from cell membrane-derived vesicles

Artificial intelligence-driven design of the assembled major cat allergen Fel d 1 to improve its spatial folding and IgE-reactivity

BackgroundCat-derived allergens are considered as one of the most common causes of allergic diseases worldwide. Fel d 1 is a major cat allergen and plays an important role in immunoglobulin E (IgE)-reaction diagnosis. However, the two separate chains of Fel d 1 exhibited lower IgE-reactivity than its complete molecule of an assembled form, which makes it difficult to efficiently prepare and limits the application of Fel d 1 in molecular diagnosis of cat allergy. MethodsWe first applied artificial intelligence (AI) based tool AlphaFold2 to build the 3-dimensional structures of Fel d 1 with different connection modes between two chains, which were evaluated by ERRAT program and were expressed in Escherichia coli. We then calculated the expression ratios of soluble form/inclusion bodies form of optimized Fel d 1. The Circular Dichroism (CD), High Performance Liquid Chromatography-Size Exclusion Chromatography (HPLC-SEC) and reducing/non-reducing SDS-PAGE were performed to characterize the folding status and dimerization of the optimized fusion Fel d 1. The improvement of specific-IgE reactivity to optimized fusion Fel d 1 was investigated by enzyme linked immunosorbent assay (ELISA). ResultsAmong several linkers, 2 × GGGGS got the highest scores, with an overall quality factor of 100. The error value of the residues around the junction of 2 × GGGGS was lower than others. It exhibited highest proportion of soluble protein than other Fel d 1 constructs with ERRAT (GGGGS, KK as well as direct fusion Fel d 1). The results of CD and HPLC-SEC showed the consistent folding and dimerization of two fused subunits between the optimized fusion Fel d 1 and previously well-defined direct fusion Fel d 1. The overall IgE-binding absorbance of optimized fusion Fel d 1 tested by ELISA was improved compared with that of the direct fusion Fel d 1. ConclusionWe firstly provided an AI-design strategy to optimize the Fel d 1, which could spontaneously fold into its native-like structure without additional refolding process or eukaryotic folding factors. The improved IgE-binding activity and simplified preparation method could greatly facilitate it to be a robust allergen material for molecular diagnosis of cat allergy.

DiffBindFR: an SE(3) equivariant network for flexible protein-ligand docking.

Molecular docking, a key technique in structure-based drug design, plays pivotal roles in protein-ligand interaction modeling, hit identification and optimization, in which accurate prediction of protein-ligand binding mode is essential. Conventional docking approaches perform well in redocking tasks with known protein binding pocket conformation in the complex state. However, in real-world docking scenario without knowing the protein binding conformation for a new ligand, accurately modeling the binding complex structure remains challenging as flexible docking is computationally expensive and inaccurate. Typical deep learning-based docking methods do not explicitly consider protein side chain conformations and fail to ensure the physical plausibility and detailed atomic interactions. In this study, we present DiffBindFR, a full-atom diffusion-based flexible docking model that operates over the product space of ligand overall movements and flexibility and pocket side chain torsion changes. We show that DiffBindFR has higher accuracy in producing native-like binding structures with physically plausible and detailed interactions than available docking methods. Furthermore, in the Apo and AlphaFold2 modeled structures, DiffBindFR demonstrates superior advantages in accurate ligand binding pose and protein binding conformation prediction, making it suitable for Apo and AlphaFold2 structure-based drug design. DiffBindFR provides a powerful flexible docking tool for modeling accurate protein-ligand binding structures.

Potency and durability of T and B cell immune responses after homologous and heterologous vector delivery of a trimer-stabilized, membrane-displayed HIV-1 clade ConC Env protein.

The generation of an HIV-1 vaccine able to induce long-lasting protective immunity remains a main challenge. Here, we aimed to modify next-generation soluble, prefusion-stabilized, close-to-native, glycan-engineered clade C gp140 envelope (Env) trimers (sC23v4 KIKO and ConCv5 KIKO) for optimal display on the cell surface following homologous or heterologous vector delivery. A combination of the following modifications scored best regarding the preservation of closed, native-like Env trimer conformation and antigenicity when using a panel of selected broadly neutralizing (bnAb) and non-neutralizing (nnAb) monoclonal antibodies for flow cytometry: i) replacing the natural cleavage site with a native flexible linker and introducing a single amino acid substitution to prevent CD4 binding (*), ii) fusing a heterologous VSV-G-derived transmembrane moiety to the gp140 C-terminus, and iii) deleting six residues proximal to the membrane. When delivering membrane-tethered sC23v4 KIKO* and ConCv5 KIKO* via DNA, VSV-GP, and NYVAC vectors, the two native-like Env trimers provide differential antigenicity profiles. Whereas such patterns were largely consistent among the different vectors for either Env trimer, the membrane-tethered ConCv5 KIKO* trimer adopted a more closed and native-like structure than sC23v4 KIKO*. In immunized mice, VSV-GP and NYVAC vectors expressing the membrane-tethered ConCv5 KIKO* administered in prime/boost combination were the most effective regimens for the priming of Env-specific CD4 T cells among all tested combinations. The subsequent booster administration of trimeric ConCv5 KIKO* Env protein preserved the T cell activation levels between groups. The evaluation of the HIV-1-specific humoral responses induced in the different immunization groups after protein boosts showed that the various prime/boost protocols elicited broad and potent antibody responses, preferentially of a Th1-associated IgG2a subclass, and that the obtained antibody levels remained high at the memory phase. In summary, we provide a feasible strategy to display multiple copies of native-like Env trimers on the cell surface, which translates into efficient priming of sustained CD4+ T cell responses after vector delivery as well as broad, potent, and sustained antibody responses following booster immunizations with the homologous, prefusion-stabilized, close-to-native ConCv5 KIKO* gp140 Env trimer.

SDBS induces multiple catalase conformations in a dose-dependent manner

Amyloid fibrils have been linked to several incurable diseases. They are long and thin fibrous proteins that self-assemble into fibrils. Small molecules can stimulate amyloid fibrillation, but the mechanism by which this happens is not well understood. This study examined how a negatively charged benzene ring containing surfactant, sodium dodecylbenzene sulphonate (SDBS), affects the fibrillation of bovine liver catalase (BLC). After SDBS treatment, BLC conformational changes were examined in vitro using turbidity, RLS kinetics, intrinsic fluorescence, ThT fluorescence, far-UV CD, and TEM. BLC in the native state was alpha-helical at pH 7.4, while it was converted to a random coil structure at pH 2.0. Far-UV CD and intrinsic fluorescence data showed that at concentrations <0.1 mM of SDBS, randomly coiled BLC assumed a native-like alpha-helical structure. However, between 0.1 and 1.0 mM SDBS, BLC was aggregated. ThT fluorescence and far-UV CD measurements showed the amyloid-like structures in the aggregated BLC. At higher SDBS concentrations (>1.0 mM) at pH 2.0, BLC again attains a native-like alpha-helical structure. It is essential for therapeutic purposes to clearly understand the process underlying surfactant- or lipid-induced fibrillation.

The role of hydrophobic interactions in the molten globule state of globular protein modulated by surfactants.

In order to highlight the role of hydrophobic interactions in the molten globule (MG) state of globular protein modulated by surfactants, the interactions of bovine α-lactalbumin (α-LA) with alkyl trimethylammonium bromides (CnTAB, n=10, 12, 14, and 16) have been studied by experimental and theoretical techniques. Isothermal titration calorimetry (ITC) showed that the enthalpy changes (ΔH) and area of the enthalpogram increased with increasing the chain length of CnTAB. The result of fluorescence, circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectrum suggested that C10TAB and C12TAB unfolded α-LA partially, C14TAB reconstructed protein with a native-like secondary structure content, and C16TAB induced an MG state α-LA. The SAXS results confirmed that the tertiary structure of α-LA was disrupted by C16TAB forming an MG state complex with a micelle-like structure even at the surfactants concentrations below CMC. As indicated by MD results, the β-domain and unstructured region(s) were involved in the MG state α-LA modulated by CnTAB. This work not only provides molecular insights into the role of hydrophobic interactions in the MG state of a globular protein but also helps understand the mechanism of preparing α-LA based biomacromolecule modulated by hydrophobic interactions.

Polyamine detergents tailored for native mass spectrometry studies of membrane proteins

Native mass spectrometry (MS) is a powerful technique for interrogating membrane protein complexes and their interactions with other molecules. A key aspect of the technique is the ability to preserve native-like structures and noncovalent interactions, which can be challenging depending on the choice of detergent. Different strategies have been employed to reduce charge on protein complexes to minimize activation and preserve non-covalent interactions. Here, we report the synthesis of a class of polyamine detergents tailored for native MS studies of membrane proteins. These detergents, a series of spermine covalently attached to various alkyl tails, are exceptional charge-reducing molecules, exhibiting a ten-fold enhanced potency over spermine. Addition of polyamine detergents to proteins solubilized in maltoside detergents results in improved, charge-reduced native mass spectra and reduced dissociation of subunits. Polyamine detergents open new opportunities to investigate membrane proteins in different detergent environments that have thwarted previous native MS studies.

Elucidating the Role of a Calcium-Binding Loop in an x-Prolyl Aminodipeptidase from Lb. helveticus.

Prolyl aminodipeptidase (PepX) is an α/β hydrolase that cleaves at penultimate N-terminal prolyl peptide bonds. The crystal structure of PepX from Lactobacillus helveticus exhibits a calcium-binding loop within the catalytic domain. The calcium-binding sequence of xDxDxDGxxD within this loop is highly conserved in PepX proteins among lactic acid bacteria, but its purpose remains unknown. Enzyme activity is not significantly affected in the presence of the metal chelator ethylenediaminetetraacetic acid (EDTA), nor in the presence of excess calcium ions. To eliminate calcium binding, D196A and D194A/D196A mutations were constructed within the conserved calcium-binding sequence motif. Enzyme activity and stability of the D196A mutant were comparable to the wild-type enzyme by colorimetric kinetic assays and protein thermal shift assays. However, the D194A/D196A mutant was inactive though it retained native-like structure and thermal stability, contradicting the EDTA and calcium titration results. This suggests calcium binding to PepX may be essential for activity.

Bovine liver catalase turns into three conformational states after exposure to an anionic surfactant.

The mechanism by which anionic surfactants promote amyloid fibril is not well understood. Here, we investigated how sodium dodecyl sulfate (SDS), a negatively charged surfactant, affects the fibrillation of the partially unfolded random-coiled bovine liver catalase (BLC) at a pH of 2.0. We used several methods, including turbidity, RLS kinetics, intrinsic fluorescence, ThT fluorescence, far-UV CD, and TEM imaging, to evaluate the conformational changes of BLC in vitro in response to SDS treatment. BLC is a multimeric protein and well folded at physiological pH but forms a random coil structure at pH 2.0. Intrinsic fluorescence and far-UV CD data showed that below 0.1mM SDS, random coiled BLC turned into a native-like structure. BLC incubated with an SDS concentration ranging from 0.1 to 2.0mM led to the formation of aggregates. The ThT fluorescence intensity was enhanced in the aggregated BLC samples (0.1-2.0mM SDS), and cross beta-sheeted structure was detected by the far UV CD measurements. BLC adopts a complete alpha-helical structure upon interacting with SDS at a more than 2.0mM concentration at pH 2.0. Understanding the mechanism of surfactant- or lipid-induced fibrillation is important for therapeutic purposes.