pH-induced Conformational Changes Research Articles

PH-induced conformational changes of lupin protein-pectin mixtures and its effect on air-water interfacial properties and foaming functionality

Lupin protein isolate (LPI) has high nutritional value and good foaming properties around neutral pH; however, its functionality becomes poor at acidic pH, due to reduced protein solubility. The addition of pectin to LPI can increase its solubility at acidic pH and hence improve protein functionality. Here, we investigated the air-water interfacial and foaming properties of LPI-pectin (1:1) mixtures at pH 3.5–7.0. We used interfacial shear and dilatational rheology, characterized the air-water interfacial microstructure with AFM of Langmuir-Blodgett films, and linked the results to the foaming properties of the LPI-pectin mixtures. Based on the phase diagram, LPI and pectin formed co-soluble mixtures at pH 6.0 and 7.0, while LPI-pectin electrostatic complexes were formed at pH 3.5 and 4.0. In the co-soluble mixtures, proteins diffused faster towards the air-water interface than the electrostatic complexes, due to smaller particle sizes of the proteins. Their air-water interfaces showed distinct differences with respect to microstructure and mechanical properties. The interfaces stabilized by co-soluble mixtures were dominated by protein aggregates, leading to weaker interfaces in response to shear and dilatational deformation, while the complexes formed thicker and denser polymeric air-water interfaces that were stiffer and more solid-like. As a result, the complex-stabilized foams were more stable than those stabilized with co-soluble mixtures. Findings from this study indicate that soluble LPI-pectin complexes formed at pH 3.5 and 4.0 were more efficient in improving interfacial and foaming properties of LPI than the co-soluble mixtures at pH 6.0 and 7.0, which can be used to tailor the properties of acid aerated products stabilized by LPI.

Impact of pH on the structure, interfacial and foaming properties of pea protein isolate: Investigation of the structure – Function relationship

This study explored the relationship between pea protein foaming properties and their structure and physicochemical properties under neutral and acidic pH. Results showed that pH modified the zeta potential, particle size and surface tension due to electrostatic changes. FT-MIR and fluorescence spectra revealed pH-induced conformational changes, exposing hydrophobic groups and increasing sulfhydryl content, promoting protein aggregation. At pH 3, the highest foaming capacity (1.273) and lowest foam expansion (6.967) were observed, associated with increased surface hydrophobicity and net charges, ideal for creating light foams with high liquid incorporation for acidic beverages or fruit-based mousses. Pea protein isolate generated stable foams with foam volume stability between 86.662 % and 94.255 %. Although neutral pH conditions showed the highest foam volume stability, their air bubbles increased in size and transitioned from spherical to polyhedral shape, suitable for visual-centric applications, like cappuccino foam and beer-head retention. Foams at pH 5 exhibited the smallest bubbles and maintained their spherical shape, enhancing drainage resistance, beneficial for whipped toppings. Strong correlations (Pearson correlation coefficient higher than 0.600) were noted between the structure, surface and foaming properties, providing crucial insights into optimizing pea protein functionality across various pH conditions, enabling the development of plant-based foamed products with tailored properties.

Biophysical characterization of chloride intracellular channel 6 (CLIC6)

Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K+) solutions, we determined that CLIC6 is more permeable to chloride-(Cl-) as compared to bromide-(Br-), fluoride-(F-), and K+ ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10μM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl- channel.

Characterization of pH-induced conformational changes in recombinant DENV NS2B-NS3pro

The increasing frequency of Dengue is a cause of severe epidemics and therefore demands strategies for effective prevention, diagnosis, and treatment. DENV-protease is being investigated as a potential therapeutic target. However, due to the flat and highly charged active site of the DENV-protease, designing orthosteric medicines is very difficult. In this study, we have done a thorough analysis of pH-dependent conformational changes in recombinantly expressed DENV protease using various spectroscopic techniques. Our spectroscopic study of DENV protease (NS2B-NS3pro) at different pH conditions gives important insights into the dynamicity of structural conformation. At physiological pH, the DENV-protease exists in a random-coiled state. Lowering the pH promotes the formation of alpha-helical and beta-sheet structures i.e. gain of secondary structure as shown by Far-UV CD. The light scattering and Thioflavin T (ThT)-binding assay proved the aggregation-prone tendency of DENV-protease at pH 4.0. Further, the confocal microscopy image intensity showed the amorphous aggregate formation of DENV protease at pH 4.0. Thus, the DENV protease acquires different conformations with changes in pH conditions. Together, these results have the potential to facilitate the design of a conformation destabilizer-based therapeutic strategy for dengue fever.

The M2 proteins of bat influenza A viruses reveal atypical features compared to conventional M2 proteins.

The influenza A virus (IAV) M2 protein has proton channel activity, which plays a role in virus uncoating and may help to preserve the metastable conformation of the IAV hemagglutinin (HA). In contrast to the highly conserved M2 proteins of conventional IAV, the primary sequences of bat IAV H17N10 and H18N11 M2 proteins show remarkable divergence, suggesting that these proteins may differ in their biological function. We, therefore, assessed the proton channel activity of bat IAV M2 proteins and investigated its role in virus replication. Here, we show that the M2 proteins of bat IAV did not fully protect acid-sensitive HA of classical IAV from low pH-induced conformational change, indicating low proton channel activity. Interestingly, the N31S substitution not only rendered bat IAV M2 proteins sensitive to inhibition by amantadine but also preserved the metastable conformation of acid-sensitive HA to a greater extent. In contrast, the acid-stable HA of H18N11 did not rely on such support by M2 protein. When mutant M2(N31S) protein was expressed in the context of chimeric H18N11/H5N1(6:2) encoding HA and NA of avian IAV H5N1, amantadine significantly inhibited virus entry, suggesting that ion channel activity supported virus uncoating. Finally, the cytoplasmic domain of the H18N11 M2 protein mediated rapid internalization of the protein from the plasma membrane leading to low-level expression at the cell surface. However, cell surface levels of H18N11 M2 protein were significantly enhanced in cells infected with the chimeric H18N11/H5N1(6:2) virus. The potential role of the N1 sialidase in arresting M2 internalization is discussed. IMPORTANCE Bat IAV M2 proteins not only differ from the homologous proteins of classical IAV by their divergent primary sequence but are also unable to preserve the metastable conformation of acid-sensitive HA, indicating low proton channel activity. This unusual feature may help to avoid M2-mediated cytotoxic effects and inflammation in bats infected with H17N10 or H18N11. Unlike classical M2 proteins, bat IAV M2 proteins with the N31S substitution mediated increased protection of HA from acid-induced conformational change. This remarkable gain of function may help to understand how single point mutations can modulate proton channel activity. In addition, the cytoplasmic domain was found to be responsible for the low cell surface expression level of bat IAV M2 proteins. Given that the M2 cytoplasmic domain of conventional IAV is well known to participate in virus assembly at the plasma membrane, this atypical feature might have consequences for bat IAV budding and egress.

Raman Spectroscopic Analysis of Highly-Concentrated Antibodies under the Acid-Treated Conditions

PurposeAntibody drugs are usually formulated as highly-concentrated solutions, which would easily generate aggregates, resulting in loss of efficacy. Although low pH increases the colloidal dispersion of antibodies, acid denaturation can be an issue. Therefore, knowing the physical properties at low pH under high concentration conditions is important.MethodsRaman spectroscopy was used to investigate pH-induced conformational changes of antibodies at 50 mg/ml. Experiments in pH 3 to 7 were performed for human serum IgG and recombinant rituximab.ResultsWe detected the evident changes at pH 3 in Tyr and Trp bands, which are the sensitive markers of intermolecular interactions. Thermal transition analysis over the pH range demonstrated that the thermal transition temperature (Tm) was highest at pH 3. Acid-treated and neutralized one showed higher Tm than that of pH 7, indicating that their extent of intermolecular interactions correlated with the Tm values. Onset temperature was clearly different between concentrated and diluted samples. Colloidal analyses confirmed the findings of the Raman analysis.ConclusionOur studies demonstrated the positive correlation between Raman analysis and colloidal information, validating as a method for evaluating antibody conformation associated with aggregation propensities.

Deciphering the alkaline stable mechanism of bacterial laccase from Bacillus pumilus by molecular dynamics simulation can improve the decolorization of textile dyes.

Laccases are considered promising tools for removing synthetic dyes from textile and tannery effluents. However, the alkaline pH in the effluents causes laccase instability, inactivation, and difficulty in its bioremediation. Based on a Bacillus pumilus ZB1 (BpLac) derived alkaline stable laccase, this study aimed to elucidate its alkaline stable mechanism at molecular level using molecular dynamics simulation. The effects of metal ions, organic solvents, and inhibitors on BpLac activity were assessed. BpLac formed more salt bridges and negatively charged surface in alkaline environment. Thereafter, pH-induced conformation changes were analyzed using GROMACS at pH 5.0 and 10.0. Among the identified residues with high fluctuation, the distance between Pro359 and Thr414 was stable at pH 10.0 but highly variable at pH 5.0. DSSP analysis suggested that BpLac formed more β-sheet and less coil at pH 10.0. Principal component analysis and free energy landscape indicated that irregular coils formed at pH 5.0 benefit for activity, while rigid α-helix and β-sheet structures formed at pH 10.0 contributed to alkaline stability. Breaking the α-helix near T1 copper center would not reduce alkaline stability but could improve dye decolorization by BpLac. Overall, these findings would advance the potential application of bacterial laccase in alkaline effluent treatment.

Influence of pH and low/high- methoxy pectin complexation on the hydrophobic binding sites of β-lactoglobulin studied by a fluorescent probe method

β-lactoglobulin(β-Lg)-polysaccharide soluble complexes formed in a specific pH range through electrostatic attraction have attracted a growing interest in the design of food-grade encapsulation systems for hydrophobic compounds, which is mainly ascribed to the ligand-binding properties of β-Lg. However, it remains unclear whether pH-induced conformational changes in β-Lg and its electrostatic complexation with anionic pectin affect their ability to bind hydrophobic compounds. Here, a fluorescent probe method was employed to provide useful insights into the field. Three solvatochromic fluorescent probes (i.e. Nile red, retinol and curcumin) were selected as representative models of hydrophobic compounds that were bound to the inner cavity or/and the outer surface of β-Lg. Binding with β-Lg or β-Lg/pectin complexes largely enhanced the fluorescence of the hydrophobic probes. Especially, the polarity difference of different binding sites on β-Lg was revealed by the fluorescence spectra of β-Lg-Nile red as a function of pH. Both Nile red and retinol were bound more favorably to β-Lg at neutral pH than at acidic pH, possibly due to the accessibility of the inner cavity in the former case. Upon acidification, the gradual reduction in fluorescence intensity of the pre-formed protein-ligands complexes (i.e. at pH 7.0) was ascribed to the dissociation between Nile red (or retinol) and the inner cavity of β-Lg. β-Lg-curcumin interactions were less affected by pH variations, suggesting curcumin mainly binding to the outer surface of β-Lg. For the three tested hydrophobic compounds, the formation of soluble complexes between β-Lg with pectin had no adverse effects on their interactions with the protein. These results may provide useful insights into the binding of hydrophobic compounds to β-Lg or β-Lg/pectin complexes. The methodology may also be extended to study the encapsulation performance of other biopolymers or particles.

From nanoparticles to crystals: one-pot programmable biosynthesis of photothermal gold structures and their use for biomedical applications

Inspired by nature, green chemistry uses various biomolecules, such as proteins, as reducing agents to synthesize metallic nanostructures. This methodology provides an alternative route to conventional harsh synthetic processes, which include polluting chemicals. Tuning the resulting nanostructure properties, such as their size and shape, is challenging as the exact mechanism involved in their formation is still not well understood. This work reports a well-controlled method to program gold nanostructures' shape, size, and aggregation state using only one protein type, mucin, as a reduction and capping material in a one-pot bio-assisted reaction. Using mucin as a gold reduction template while varying its tertiary structure via the pH of the synthesis, we demonstrate that spherical, coral-shaped, and hexagonal gold crystals can be obtained and that the size can be tuned over three orders of magnitude. This is achieved by leveraging the protein's intrinsic reducing properties and pH-induced conformational changes. The systematic study of the reaction kinetics and growth steps developed here provides an understanding of the mechanism behind this phenomenon. We further show that the prepared gold nanostructures exhibit tunable photothermal properties that can be optimized for various hyperthermia-induced antibacterial applications.

Macromolecular conformational changes in photosystem II: interaction between structure and function.

Conformational changes play an important role in the functioning of proteins and their complexes. This is also true for the pigment-protein super-complex of photosystem II (PSII). The data testify about the pH-induced macromolecular conformational changes in the water-oxidizing complex (WOC) on the donor side of PSII, the interaction between the spatial structure of WOC proteins and the distribution of cytochrome b559 redox-forms, and the electron transfer efficiency between QA and QB on the acceptor side of PSII. Changes in the protein environment near QA and QB can be observed after the removal of the bicarbonate ion associated with non-heme Fe or after the addition of herbicides binding to the QB site, which results in the suppression of the electron transfer in this site. The "locking" of the de novo assembled PSII in an inactive state until WOC activation is also accompanied by strong structural perturbations on the PSII acceptor and donor sides with the participation of Psb28 and Psb27 proteins. The triggers for degradation and replacement of damaged PSII proteins are structural changes induced by their oxidative modification and aggregation. Macromolecular changes in the antenna proteins underlie the activation of photoprotective non-photochemical quenching, which are induced by protonation of the lumenal residues of PsbS or/and Lhcsr3, as well as the phosphorylation of antenna proteins. Besides this, many smaller-scale conformational changes may occur in PSII. This review summarizes current knowledge about the possible conformational changes in proteins in the PSII super-complex and describes their proposed influence on PSII function.

Synthesis of Analogues of Arbidol and Their Anti-viral Studies Towards Chikungunya Virus

The fusion of virus and endosome membranes is an essential early stage in chikungunya virus infection. The low pH-induced conformational change which promotes the fusogenic activity of the haemagglutinin (HA) is thus an attractive target as an antiviral strategy. The anti-chikungunya drug, arbidol is representative of a class of antivirals which inhibits HA-mediated membrane fusion by increasing the acid stability of the HA. In this study two series of indole derivatives structurally related to Arbidol were designed and synthesized to further probe the foundation of its antiviral activity and develop the basis for a structure–activity relationship (SAR). Ethyl 5-(hydroxymethyl)-1-methyl-2-(phenysulphanylmethyl)-1H-indole-3-carboxylate was identified as one of the most potent inhibitors and more potent than Arbidol against certain subtypes of chikungunya viruses.

Generation of False-Positive SARS-CoV-2 Antigen Results with Testing Conditions outside Manufacturer Recommendations: A Scientific Approach to Pandemic Misinformation.

ABSTRACTAntigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. However, misleading demonstrations of the Abbott Panbio coronavirus disease 2019 (COVID-19) Ag-RDT on social media claimed that SARS-CoV-2 antigen could be detected in municipal water and food products. To offer a scientific rebuttal to pandemic misinformation and disinformation, this study explored the impact of using the Panbio SARS-CoV-2 assay with conditions falling outside manufacturer recommendations. Using Panbio, various water and food products, laboratory buffers, and SARS-CoV-2-negative clinical specimens were tested with and without manufacturer buffer. Additional experiments were conducted to assess the role of each Panbio buffer component (tricine, NaCl, pH, and Tween 20) as well as the impact of temperature (4°C, 20°C, and 45°C) and humidity (90%) on assay performance. Direct sample testing (without the kit buffer) resulted in false-positive signals resembling those obtained with SARS-CoV-2 positive controls tested under proper conditions. The likely explanation of these artifacts is nonspecific interactions between the SARS-CoV-2-specific conjugated and capture antibodies, as proteinase K treatment abrogated this phenomenon, and thermal shift assays showed pH-induced conformational changes under conditions promoting artifact formation. Omitting, altering, and reverse engineering the kit buffer all supported the importance of maintaining buffering capacity, ionic strength, and pH for accurate kit function. Interestingly, the Panbio assay could tolerate some extremes of temperature and humidity outside manufacturer claims. Our data support strict adherence to manufacturer instructions to avoid false-positive SARS-CoV-2 Ag-RDT reactions, otherwise resulting in anxiety, overuse of public health resources, and dissemination of misinformation.IMPORTANCE With the Panbio severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test being deployed in over 120 countries worldwide, understanding conditions required for its ideal performance is critical. Recently on social media, this kit was shown to generate false positives when manufacturer recommendations were not followed. While erroneous results from improper use of a test may not be surprising to some health care professionals, understanding why false positives occur can help reduce the propagation of misinformation and provide a scientific rebuttal for these aberrant findings. This study demonstrated that the kit buffer’s pH, ionic strength, and buffering capacity were critical components to ensure proper kit function and avoid generation of false-positive results. Typically, false positives arise from cross-reacting or interfering substances; however, this study demonstrated a mechanism where false positives were generated under conditions favoring nonspecific interactions between the two antibodies designed for SARS-CoV-2 antigen detection. Following the manufacturer instructions is critical for accurate test results.

A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity.

Thrombin activation of C5 connects thrombosis to inflammation. Complement research in whole blood ex vivo necessitates anticoagulation, which potentially interferes with the inflammatory modulation by thrombin. We challenged the concept of thrombin as an activator of native C5 by analyzing complement activation and C5 cleavage in human whole blood anticoagulated with Gly-Pro-Arg-Pro (GPRP), a peptide targeting fibrin polymerization downstream of thrombin, allowing complete endogenous thrombin generation. GPRP dose-dependently inhibited coagulation but allowed for platelet activation in accordance with thrombin generation. Spontaneous and bacterial-induced complement activation by Escherichia coli and Staphylococcus aureus, analyzed at the level of C3 and C5, were similar in blood anticoagulated with GPRP and the thrombin inhibitor lepirudin. In the GPRP model, endogenous thrombin, even at supra-physiologic concentrations, did not cleave native C5, despite efficiently cleaving commercially sourced purified C5 protein, both in buffer and when added to C5-deficient serum. In normal serum, only exogenously added, commercially sourced C5 was cleaved, whereas the native plasma C5 remained intact. Crucially, affinity-purified C5, eluted under mild conditions using an MgCl2 solution, was not cleaved by thrombin. Acidification of plasma to pH ≤ 6.8 by hydrochloric or lactic acid induced a C5 antigenic change, nonreversible by pH neutralization, that permitted cleavage by thrombin. Circular dichroism on purified C5 confirmed the structural change during acidification. Thus, we propose that pH-induced conformational change allows thrombin-mediated cleavage of C5 and that, contrary to previous reports, thrombin does not cleave plasma C5 in its native form, suggesting that thrombin cleavage of C5 may be restricted to certain pathophysiological conditions.

Stability of HA2 Prefusion Structure and pH-Induced Conformational Changes in the HA2 Domain of H3N2 Hemagglutinin.

Influenza hemagglutinin is the fusion protein that mediates fusion of the viral and host membranes through a large conformational change upon acidification in the developing endosome. The "spring-loaded" model has long been used to describe the mechanism of hemagglutinin and other type 1 viral glycoproteins. This model postulates a metastable conformation of the HA2 subunit, caged from adopting a lower-free energy conformation by the HA1 subunit. Here, using a combination of biochemical and spectroscopic methods, we study a truncated construct of HA2 (HA2*, lacking the transmembrane domain) recombinantly expressed in Escherichia coli as a model for HA2 without the influence of HA1. Our data show that HA2* folds into a conformation like that of HA2 in full length HA and forms trimers. Upon acidification, HA2* undergoes a conformational change that is consistent with the change from pre- to postfusion HA2 in HA. This conformational change is fast and occurs on a time scale that is not consistent with aggregation. These results suggest that the prefusion conformation of HA2 is stable and the change to the postfusion conformation is due to protonation of HA2 itself and not merely uncaging by HA1.

PH-induced changes in β-casomorphin 7 structure studied by 1H nuclear magnetic resonance and Fourier-transform infrared spectroscopy

β-Casomorphins are opioid peptides that can be liberated from β-casein during digestion, with some suggested effects on human physiology linked to their properties. In this study, the structural behaviour of β-casomorphin 7 (BCM7; Tyr–Pro–Phe–Pro–Gly–Pro–Ile) was studied at pH 2.3 and 6.7 using 1H nuclear magnetic resonance (1H NMR) and Fourier-transform infrared (FTIR) spectroscopy. Cis–trans isomerism involving the Tyr1–Pro2, Phe3–Pro4 and Gly5–Pro6 bonds of BCM7 was observed. Cis and trans isomers of Tyr1–Pro2 and Phe3–Pro4 and trans isomers for Gly5–Pro6 were observed at both pH 6.7 and 2.3 but cis isomers of Gly5–Pro6 were only observed at pH 2.3 and not at pH 6.7. These pH-induced conformational changes at the Gly5–Pro6 bond of BCM7 may play important role in enzymatic release of this peptide from the parent protein β-casein under gastro-intestinal conditions, as well as further cleavage of BCM7 into smaller peptides.

Development of mouse monoclonal antibody for detecting hemagglutinin of avian influenza A(H7N9) virus and preventing virus infection

Many cases of avian influenza A(H7N9) virus infection in humans have been reported since its first emergence in 2013. The disease is of concern because most patients have become severely ill with roughly 30% mortality rate. Because the threat in public health caused by H7N9 virus remains high, advance preparedness is essentially needed. In this study, the recombinant H7N9 hemagglutinin (HA) was expressed in insect cells and purified for generation of two monoclonal antibodies, named F3-2 and 1C6B. F3-2 can only recognize the H7N9 HA without having cross-reactivity with HA proteins of H1N1, H3N2, H5N1, and H7N7. 1C6B has the similar specificity with F3-2, but 1C6B can also bind to H7N7 HA. The binding epitope of F3-2 is mainly located in the region of H7N9 HA(299–307). The binding epitope of 1C6B is located in the region of H7N9 HA(489–506). F3-2 and 1C6B could not effectively inhibit the hemagglutination activity of H7N9 HA. However, F3-2 can prevent H7N9 HA from trypsin cleavage and can bind to H7N9 HA which has undergone pH-induced conformational change. F3-2 also has the ability of binding to H7N9 viral particles and inhibiting H7N9 virus infection to MDCK cells with the IC50 value of 22.18 μg/mL. In addition, F3-2 and 1C6B were utilized for comprising a lateral flow immunochromatographic test strip for specific detection of H7N9 HA.Key points• Two mouse monoclonal antibodies, F3-2 and 1C6B, were generated for recognizing the novel binding epitopes in H7N9 HA.• F3-2 can prevent H7N9 HA from trypsin cleavage and inhibit H7N9 virus infection to MDCK cells.• F3-2 and 1C6B were developed as a lateral flow immunochromatographic test for specific detection of H7N9 HA.

PH-Induced Conformational Changes of Human Bocavirus Capsids.

Human bocavirus 1 (HBoV1) and HBoV2-4 infect children and immunocompromised individuals, resulting in respiratory and gastrointestinal infections, respectively. Using cryo-electron microscopy and image reconstruction, the HBoV2 capsid structure was determined to 2.7 Å resolution at pH 7.4 and compared to the previously determined HBoV1, HBoV3, and HBoV4 structures. Consistent with previous findings, surface variable region (VR) III of the capsid protein VP3, proposed as a host tissue-tropism determinant, was structurally similar among the gastrointestinal strains HBoV2-4, but differed from HBoV1 with its tropism for the respiratory tract. Towards understanding the entry and trafficking properties of these viruses, HBoV1 and HBoV2 were further analyzed as species representatives of the two HBoV tropisms. Their cell surface glycan-binding characteristics were analyzed, and capsid structures determined to 2.5-2.7 Å resolution at pH 5.5 and 2.6, conditions normally encountered during infection. The data showed that glycans with terminal sialic acid, galactose, GlcNAc or heparan sulfate moieties do not facilitate HBoV1 or HBoV2 cellular attachment. With respect to trafficking, conformational changes common to both viruses were observed at low pH conditions localized to the VP N-terminus under the 5-fold channel, in the surface loops VR-I and VR-V and specific side-chain residues such as cysteines and histidines. The 5-fold conformational movements provide insight into the potential mechanism of VP N-terminal dynamics during HBoV infection and side-chain modifications highlight pH-sensitive regions of the capsid.IMPORTANCE Human bocaviruses (HBoVs) are associated with disease in humans. However, the lack of an animal model and a versatile cell culture system to study their life cycle limits the ability to develop specific treatments or vaccines. This study presents the structure of HBoV2, at 2.7 Å resolution, determined for comparison to the existing HBoV1, HBoV3, and HBoV4 structures, to enable the molecular characterization of strain and genus-specific capsid features contributing to tissue tropism and antigenicity. Furthermore, HBoV1 and HBoV2 structures determined under acidic conditions provide insight into capsid changes associated with endosomal and gastrointestinal acidification. Structural rearrangements of the capsid VP N-terminus, at the base of the 5-fold channel, demonstrate a disordering of a "basket" motif as pH decreases. These observations begin to unravel the molecular mechanism of HBoV infection and provide information for control strategies.

A pH-Induced Reversible Conformational Switch Able to Control the Photocurrent Efficiency in a Peptide Supramolecular System.

External stimuli are potent tools that Nature uses to control protein function and activity. For instance, during viral entry and exit, pH variations are known to trigger large protein conformational changes. In Nature, also the electron transfer (ET) properties of ET proteins are influenced by pH-induced conformational changes. In this work, a pH-controlled, reversible 310 -helix to α-helix conversion (from acidic to highly basic pH values and vice versa) of a peptide supramolecular system built on a gold surface is described. The effect of pH on the ability of the peptide SAM to generate a photocurrent was investigated, with particular focus on the effect of the pH-induced conformational change on photocurrent efficiency. The films were characterized by electrochemical and spectroscopic techniques, and were found to be very stable over time, also in contact with a solution. They were also able to generate current under illumination, with an efficiency that is the highest recorded so far with biomolecular systems.

Acidic pH-Induced Conformational Changes in Chikungunya Virus Fusion Protein E1: a Spring-Twisted Region in the Domain I-III Linker Acts as a Hinge Point for Swiveling Motion of Domains.

Chikungunya virus (CHIKV), a mosquito-transmitted alphavirus, enters a cell through endocytosis, followed by viral and cell membrane fusion. The fusion protein, E1, undergoes an acid pH-induced pre- to postfusion conformation change during membrane fusion. As part of the conformation change, E1 dissociates from the receptor-binding protein, E2, and swivels its domains I and II over domain III to form an extended intermediate and then eventually to form a postfusion hairpin homotrimer. In this study, we tested if the domain I-III linker acts as a "hinge" for the swiveling motion of E1 domains. We found a conserved spring-twisted structure in the linker, stabilized by a salt bridge between a conserved arginine-aspartic acid pair, as a "hinge point" for domain swiveling. Molecular dynamics (MD) simulation of the CHIKV E1 or E2-E1 structure predicted that the spring-twisted region untwists at pH 5.5. Corroborating the prediction, introduction of a "cystine staple" at the hinge point, replacing the conserved arginine-aspartic acid pair with cysteine residues, resulted in loss of fusion activity of E1. MD simulation also predicted domain I-III swiveling at acidic pH. We tested if breaking the His 331-Lys 16 H bond between domains I and III, seen only in the prefusion conformation, is important for domain swiveling. When domains I and III are "stapled" by introducing a disulfide bond in between, E1 showed loss of fusion activity, implying that domain I and III dissociation is a critical acid pH-induced step in membrane fusion. However, replacement of His 331 with an acidic residue did not affect the pH threshold for fusion, suggesting His 331 is not an acid-sensing residue.IMPORTANCEAedes mosquito-transmitted viruses such as the Zika, dengue, and chikungunya viruses have spread globally. CHIKV, similar to many other enveloped viruses, enters cells in sequential steps: step 1 involves receptor binding followed by endocytosis, and step 2 involves viral-cell membrane fusion in the endocytic vesicle. The viral envelope surface protein, E1, performs membrane fusion. E1 is triggered to undergo conformational changes by acidic pH of the maturing endosome. Different domains of E1 rearrange during the pre- to postfusion conformation change. Using in silico analysis of the E1 structure and different biochemical experiments, we explained a structural mechanism of key conformational changes in E1 triggered by acidic pH. We noted two important structural changes in E1 at acidic pH. In the first, a spring-twisted region in a loop connecting two domains (I and III) untwists, bringing a swiveling motion of domains on each other. In the second, breaking of interactions between domains I and III and domain separation are required for membrane fusion. This knowledge will help devise new therapeutic strategies to block conformation changes in E1 and thus viral entry.

PH-induced conformational changes and interfacial dilatational rheology of soy protein isolated/soy hull polysaccharide complex and its effects on emulsion stabilization

In this study, the effects of conformational conversion and interfacial dilatational rheology of soy protein isolated (SPI)/soy hull polysaccharide (SHP) on physical stability of oil-in-water (O/W) emulsions were investigated at pH 3.0–9.0. The results showed that SHP enhanced the physico-chemical stability of SPI emulsions at pH 3.0–9.0. At pH 5.0, SPI/SHP emulsions that have a smaller particle size, interfacial tension, and a higher electrostatic repulsion was developed. FTIR and Raman spectroscopy indicated that the secondary structures of the α-helix and random coil of SPI/SHP complexes changed with pH. SHP decreased the interaction between proteins adsorbed at the oil-water interface and the SPI/SHP complex presented a stronger interaction at pH 5.0. The electrostatic, hydrophobic, and hydrogen bonding interactions played an essential role in the polar or hydrophobic environment of the tryptophan and tyrosine residues inside protein molecules. These findings indicated that the SPI/SHP complex shows potential for commercial application in the production of novel functional emulsions.