Exploring the early stages of the pH‐induced conformational change of influenza hemagglutinin

Hemagglutinin (HA) mediates the membrane fusion process of influenza virus through its pH‐induced conformational change. However, it remains challenging to study its structure reorganization pathways in atomic details. Here, we first applied continuous constant pH molecular dynamics approach to predict the pK_a values of titratable residues in H2 subtype HA. The calculated net‐charges in HA1 globular heads increase from 0e (pH 7.5) to +14e (pH 4.5), indicating that the charge repulsion drives the detrimerization of HA globular domains. In HA2 stem regions, critical pH sensors, such as Glu103₂, His18₁, and Glu89₁, are identified to facilitate the essential structural reorganizations in the fusing pathways, including fusion peptide release and interhelical loop transition. To probe the contribution of identified pH sensors and unveil the early steps of pH‐induced conformational change, we carried out conventional molecular dynamics simulations in explicit water with determined protonation state for each titratable residue in different environmental pH conditions. Particularly, energy barriers involving previously uncharacterized hydrogen bonds and hydrophobic interactions are identified in the fusion peptide release pathway. Nevertheless, comprehensive comparisons across HA family members indicate that different HA subtypes might employ diverse pH sensor groups along with different fusion pathways. Finally, we explored the fusion inhibition mechanism of antibody CR6261 and small molecular inhibitor TBHQ, and discovered a novel druggable pocket in H2 and H5 subtypes. Our results provide the underlying mechanism for the pH‐driven conformational changes and also novel insight for anti‐flu drug development. Proteins 2014; 82:2412–2428. © 2014 Wiley Periodicals, Inc.     

Osmolyte‐induced conformational changes in the Hsp90 molecular chaperone

Osmolytes are small molecules that play a central role in cellular homeostasis and the stress response by maintaining protein thermodynamic stability at controlled levels. The underlying physical chemistry that describes how different osmolytes impact folding free energy is well understood, however little is known about their influence on other crucial aspects of protein behavior, such as native‐state conformational changes. Here we investigate this issue with the Hsp90 molecular chaperone, a large dimeric protein that populates a complex conformational equilibrium. Using small angle X‐ray scattering we observe dramatic osmolyte‐dependent structural changes within the native ensemble. The degree to which different osmolytes affect the Hsp90 conformation strongly correlates with thermodynamic metrics of their influence on stability. This observation suggests that the well‐established osmolyte principles that govern stability also apply to large‐scale conformational changes, a proposition that is corroborated by structure‐based fitting of the scattering data, surface area comparisons and m‐value analysis. This approach shows how osmolytes affect a highly cooperative open/closed structural transition between two conformations that differ by a domain‐domain interaction. Hsp90 adopts an additional ligand‐specific conformation in the presence of ATP and we find that osmolytes do not significantly affect this conformational change. Together, these results extend the scope of osmolytes by suggesting that they can maintain protein conformational heterogeneity at controlled levels using similar underlying principles that allow them to maintain protein stability; however the relative impact of osmolytes on different structural states can vary significantly.     

LumenP--a neural network predictor for protein localization in the thylakoid lumen

We report the development of LumenP, a new neural network-based predictor for the identification of proteins targeted to the thylakoid lumen of plant chloroplasts and prediction of their cleavage sites. When used together with the previously developed TargetP predictor, LumenP reaches a significantly better performance than what has been recorded for previous attempts at predicting thylakoid lumen location, mostly due to a lower false positive rate. The combination of TargetP and LumenP predicts around 1.5%-3% of all proteins encoded in the genomes of Arabidopsis thaliana and Oryza sativa to be located in the lumen of the thylakoid.     

Pressure‐induced conformational switch of an interfacial protein

A special class of proteins adopts an inactive conformation in aqueous solution and activates at an interface (such as the surface of lipid droplet) by switching their conformations. Lipase, an essential enzyme for breaking down lipids, serves as a model system for studying such interfacial proteins. The underlying conformational switch of lipase induced by solvent condition is achieved through changing the status of the gated substrate‐access channel. Interestingly, a lipase was also reported to exhibit pressure activation, which indicates it is drastically active at high hydrostatic pressure. To unravel the molecular mechanism of this unusual phenomenon, we examined the structural changes induced by high hydrostatic pressures (up to 1500 MPa) using molecular dynamics simulations. By monitoring the width of the access channel, we found that the protein undergoes a conformational transition and opens the access channel at high pressures (>100 MPa). Particularly, a disordered amphiphilic α5 region of the protein becomes ordered at high pressure. This positive correlation between the channel opening and α5 ordering is consistent with the early findings of the gating motion in the presence of a water–oil interface. Statistical analysis of the ensemble of conformations also reveals the essential collective motions of the protein and how these motions contribute to gating. Arguments are presented as to why heightened sensitivity to high‐pressure perturbation can be a general feature of switchable interfacial proteins. Further mutations are also suggested to validate our observations. Proteins 2016; 84:820–827. © 2016 Wiley Periodicals, Inc.     

Improving the analysis of NMR spectra tracking pH‐induced conformational changes: Removing artefacts of the electric field on the NMR chemical shift

pH‐induced chemical shift perturbations (CSPs) can be used to study pH‐dependent conformational transitions in proteins. Recently, an elegant principal component analysis (PCA) algorithm was developed and used to study the pH‐dependent structural transitions in bovine β‐lactoglobulin (βLG) by analyzing its NMR pH‐titration spectra. Here, we augment this analysis method by filtering out changes in the NMR chemical shift that stem from effects that are electrostatic in nature. Specifically, we examine how many CSPs can be explained by purely electrostatic effects arising from titrational events in βLG. The results show that around 20% of the amide nuclei CSPs in βLG originate exclusively from “through‐space” electric field effects. A PCA of NMR data where electric field artefacts have been removed gives a different picture of the pH‐dependent structural transitions in βLG. The method implemented here is well suited to be applied on a whole range of proteins, which experience at least one pH‐dependent conformational change. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Silver‐induced conformational changes of polypeptides: a CD study

The role of silver ions in various pathologies, as well as their effect on peptide conformation and properties are less understood. Consequently, we synthesized several peptides with various residues in their sequence to investigate silver‐induced conformational changes at various pH values by Circular Dichroism spectroscopy. Uniquely, the glycine‐based, histidine‐containing peptide showed a severe change from a random coil and β‐turn conformation to large α‐helices during silver binding. When comparing the effect of silver ions on the conformation of bradykinin a similar tendency was found. Besides, silver ions reduced the amyloid‐β peptide tendency to aggregation. Our results suggest a specific and protective role for silver ions in brain pathologies, which is related to their high affinity toward physiologically and pharmacologically active peptides. Fourier transform infrared spectroscopy studies as well as the mass spectrometric ones support our conclusions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Immobility of phycobilins in the thylakoid lumen of a cryptophyte suggests that protein diffusion in the lumen is very restricted

The thylakoid lumen is an important photosynthetic compartment which is the site of key steps in photosynthetic electron transport. The fluidity of the lumen could be a major constraint on photosynthetic electron transport rates. We used Fluorescence Recovery After Photobleaching in cells of the cryptophyte alga Rhodomonas salina to probe the diffusion of phycoerythrin in the lumen and chlorophyll complexes in the thylakoid membrane. In neither case was there any detectable diffusion over a timescale of several minutes. This indicates very restricted phycoerythrin mobility. This may be a general feature of protein diffusion in the thylakoid lumen.     

pH‐dependent conformational dynamics of beta‐secretase 1: A molecular dynamics study

Beta‐secretase 1 (BACE‐1) is an aspartyl protease implicated in the overproduction of β‐amyloid fibrils responsible for Alzheimer disease. The process of β‐amyloid genesis is known to be pH dependent, with an activity peak between solution pH of 3.5 and 5.5. We have studied the pH‐dependent dynamics of BACE‐1 to better understand the pH dependent mechanism. We have implemented support for graphics processor unit (GPU) accelerated constant pH molecular dynamics within the AMBER molecular dynamics software package and employed this to determine the relative population of different aspartyl dyad protonation states in the pH range of greatest β‐amyloid production, followed by conventional molecular dynamics to explore the differences among the various aspartyl dyad protonation states. We observed a difference in dynamics between double‐protonated, mono‐protonated, and double‐deprotonated states over the known pH range of higher activity. These differences include Tyr 71‐aspartyl dyad proximity and active water lifetime. This work indicates that Tyr 71 stabilizes catalytic water in the aspartyl dyad active site, enabling BACE‐1 activity.     

Calcium-dependent conformational change and thermal stability of the isolated PsbO protein detected by FTIR spectroscopy

The structure and function of the photosystem II PsbO extrinsic protein is under intense research, being an essential part of the biomolecular engine that carries out water oxidation and oxygen production. This paper presents a structural analysis of the isolated PsbO protein by FTIR spectroscopy, reporting detailed secondary structure quantification and changes in the secondary structure content of the protein attributed to the effect of calcium (Ca(2+)). Measurements in H(2)O and D(2)O have allowed us to see the effect of calcium on the conformation of the protein. The results indicate that (i) the protein presents a major content of beta-structure (i.e., beta-sheet, beta-strands, beta-turns) as detected by the infrared bands at 1624-1625, 1678-1679, 1688-1689 cm(-1), which account for about 38% in water and 33% in heavy water, in the presence of calcium; and (ii) the amount of this beta-structure fraction increases 7-10% in the absence of calcium, with a concomitant decrease in loops and nonordered structure. The thermal denaturation profile of the protein in the presence of calcium showed low stability with T(m) approximately 56 degrees C. This profile also shows a second phase of denaturation above 60 degrees C and the appearance of aggregation signals above 70 degrees C. Our observations indicate that calcium is able to modify the conformation of the protein at least in solution and confirm that PsbO is mainly a beta-protein where beta-sheet is the major ordered secondary structure element of the protein core.     

Chloroplast thylakoid protein changes induced by low growth temperature in maize revealed by immunocytology

Tissue-specific effects of low growth temperature on maize chloroplast thylakoid protein accumulation were analysed using immunocytology. Sections of leaves from plants grown at 25 and 14°C were probed with antibodies to specific chloroplast thylakoid proteins from the four major protein multisubunit complexes of the thylakoid membrane followed by fluorescein-conjugated goat anti-rabbit antibodies. At a normal growth temperature of 25°C, the 32 kDa D1 protein of the photosystem II reaction centre and the 33 kDa protein of the extrinsic oxygen-evolving complex of photosystem II are both accumulated to a greater degree in the mesophyll than in the bundle sheath chloroplasts. In contrast, subunit II of photosystem I, cytochrome f and the α- and β-subunits of ATP synthetase are predominant in the bundle sheath thylakoids at 25°C. A striking difference between the 25°C-grown and the 14°C-grown leaf tissue was the presence in the latter of (20–30%) cells whose chloroplasts apparently completely lack several of the thylakoid proteins. In plants grown at 14°C, the accumulation of the 33 kDa protein of the extrinsic oxygen-evolving complex of photosystem II was apparently unchanged, but other thylakoid proteins showed a significant reduction. The uneven distribution of proteins between the bundle sheath and mesophyll chloroplasts observed at 25°C was also maintained at 14°C. Reduction in the fluorescence at 14°C was manifested either as an overall reduction in the diffuse fluorescence across the chloroplast profiles or less frequently as a reduction to small discrete bodies of intense fluorescence. The significance of these results to low-temperature-induced reduction in the photosynthetic productivity of maize is discussed.     

Structure of HIV‐1 reverse transcriptase/d4TTP complex: Novel DNA cross‐linking site and pH‐dependent conformational changes

Stavudine (d4T, 2′,3′‐didehydro‐2′,3′‐dideoxythymidine) was one of the first chain‐terminating nucleoside analogs used to treat HIV infection. We present the first structure of the active, triphosphate form of d4T (d4TTP) bound to a catalytic complex of HIV‐1 RT/dsDNA template‐primer. We also present a new strategy for disulfide (S–S) chemical cross‐linking between N⁶ of a modified adenine at the second overhang base to I63C in the fingers subdomain of RT. The cross‐link site is upstream of the duplex‐binding region of RT, however, the structure is very similar to published RT structures with cross‐linking to Q258C in the thumb, which suggests that cross‐linking at either site does not appreciably perturb the RT/DNA structures. RT has a catalytic maximum at pH 7.5. We determined the X‐ray structures of the I63C‐RT/dsDNA/d4TTP cross‐linked complexes at pH 7, 7.5, 8, 8.5, 9, and 9.5. We found small (~0.5 Å), pH‐dependent motions of the fingers subdomain that folds in to form the dNTP‐binding pocket. We propose that the pH‐activity profile of RT relates to this motion of the fingers. Due to side effects of neuropathy and lipodystrophy, use of d4T has been stopped in most countries, however, chemical modification of d4T might lead to the development of a new class of nucleoside analogs targeting RNA and DNA polymerases.     

Molecular dynamics simulation of the phosphorylation‐induced conformational changes of a tau peptide fragment

Aggregation of the microtubule associated protein tau (MAPT) within neurons of the brain is the leading cause of tauopathies such as Alzheimer's disease. MAPT is a phospho‐protein that is selectively phosphorylated by a number of kinases in vivo to perform its biological function. However, it may become pathogenically hyperphosphorylated, causing aggregation into paired helical filaments and neurofibrillary tangles. The phosphorylation induced conformational change on a peptide of MAPT (htau_225?250) was investigated by performing molecular dynamics simulations with different phosphorylation patterns of the peptide (pThr231 and/or pSer235) in different simulation conditions to determine the effect of ionic strength and phosphate charge. All phosphorylation patterns were found to disrupt a nascent terminal β‐sheet pattern (²²⁶VAVVR²³⁰ and ²⁴⁴QTAPVP²⁴⁹), replacing it with a range of structures. The double pThr231/pSer235 phosphorylation pattern at experimental ionic strength resulted in the best agreement with NMR structural characterization, with the observation of a transient α‐helix (²³⁹AKSRLQT²⁴⁵). PPII helical conformations were only found sporadically throughout the simulations. Proteins 2014; 82:1907–1923. © 2014 Wiley Periodicals, Inc.     

RNA and CuCl2 induced conformational changes of the recombinant ovine prion protein

Prion diseases are a group of neurodegenerative illnesses caused by conformational conversion of benign, α-helix rich cellular prion protein (PrP^C) into the highly stable, β-sheet rich scrapie prion protein (PrP^Sc) isoform. To date, the role of RNA on the conformational conversion of ovine prion protein in vitro remains unknown. To examine the effect of the interaction between RNA and PrP^C, conformations of recombinant ovine prion protein PrP^23–256 (OvPrP^23–256) binding various concentrations of RNA were analyzed by circular dichroism (CD) spectrum. The results indicated that the conformational conversion of OvPrP^23–256 was triggered by RNA with a decrease in α-helix content and increase in β-sheet. Moreover, the conformation of OvPrP^23–256 interacting with both RNA and CuCl₂ was also examined by CD spectrum, which showed that α-helix content decreased while β-sheet increased dramatically. Proteinase K digestion assay disclosed that the recombinant ovine PrP^C acquired PK resistance after RNA and/or Cu²⁺ treatment. It confirmed that the RNA/Cu²⁺ treatment in vitro altered the biochemical properties of ovine PrP^C. The implication of this finding, with respect to PrP^Sc, is that a dysfunctional state of a normal physiological process possibly facilitates diseases. The information gained from this study may provide useful approaches to study the pathogenesis of prion diseases.     

Investigation of the pH‐dependence of dye‐doped protein–protein interactions

Proteins can dramatically change their conformation under environmental conditions such as temperature and pH. In this context, Glycoprotein's conformational determination is challenging. This is due to the variety of domains which contain rich chemical characters existing within this complex. Here we demonstrate a new, straightforward and efficient technique that uses the pH‐dependent properties of dyes‐doped Pig Gastric Mucin (PGM) for predicting and controlling protein–protein interaction and conformation. We utilize the PGM as natural host matrix which is capable of dynamically changing its conformational shape and adsorbing hydrophobic and hydrophilic dyes under different pH conditions and investigate and control the fluorescent properties of these composites in solution. It is shown at various pH conditions, a large variety of light emission from these complexes such as red, green and white is obtained. This phenomenon is explained by pH‐dependent protein folding and protein–protein interactions that induce different emission spectra which are mediated and controlled by means of dye–dye interactions and surrounding environment. This process is used to form the technologically challenging white light‐emitting liquid or solid coating for LED devices.     

A pH‐ and ionic strength‐dependent conformational change in the neck region regulates DNGR‐1 function in dendritic cells

DNGR‐1 is receptor expressed by certain dendritic cell (DC) subsets and by DC precursors in mouse. It possesses a C‐type lectin‐like domain (CTLD) followed by a poorly characterized neck region coupled to a transmembrane region and short intracellular tail. The CTLD of DNGR‐1 binds F‐actin exposed by dead cell corpses and causes the receptor to signal and potentiate cross‐presentation of dead cell‐associated antigens by DCs. Here, we describe a conformational change that occurs in the neck region of DNGR‐1 in a pH‐ and ionic strength‐dependent manner and that controls cross‐presentation of dead cell‐associated antigens. We identify residues in the neck region that, when mutated, lock DNGR‐1 in one of the two conformational states to potentiate cross‐presentation. In contrast, we show that chimeric proteins in which the neck region of DNGR‐1 is replaced by that of unrelated C‐type lectin receptors fail to promote cross‐presentation. Our results suggest that the neck region of DNGR‐1 is an integral receptor component that senses receptor progression through the endocytic pathway and has evolved to maximize extraction of antigens from cell corpses, coupling DNGR‐1 function to its cellular localization.     

A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen.

We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single-copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper and a potential phosphatase-binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non-appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light-mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.     

Osmolyte-induced changes in protein conformational equilibria

Examining solute-induced changes in protein conformational equilibria is a long-standing method for probing the role of water in maintaining protein stability. Interpreting the molecular details governing the solute-induced effects, however, remains controversial. We present experimental and theoretical data for osmolyte-induced changes in the stabilities of the A and N states of yeast iso-1-ferricytochrome c. Using polyol osmolytes of increasing size, we observe that osmolytes alone induce A-state formation from acid-denatured cytochrome c and N state formation from the thermally denatured protein. The stabilities of the A and N states increase linearly with osmolyte concentration. Interestingly, osmolytes stabilize the A state to a greater degree than the N state. To interpret the data, we divide the free energy for the reaction into contributions from nonspecific steric repulsions (excluded volume effects) and from binding interactions. We use scaled particle theory (SPT) to estimate the free energy contributions from steric repulsions, and we estimate the contributions from water-protein and osmolyte-protein binding interactions by comparing the SPT calculations to experimental data. We conclude that excluded volume effects are the primary stabilizing force, with changes in water-protein and solute-protein binding interactions making favorable contributions to stability of the A state and unfavorable contributions to the stability of the N state. The validity of our interpretation is strengthened by analysis of data on osmolyte-induced protein stabilization from the literature, and by comparison with other analyses of solute-induced changes in conformational equilibria.     

Retinol-binding protein in human serum: conformational changes induced by retinoic acid binding

Polyacrylamide gel electrophoresis and isoelectrofocusing followed by immunoblotting technique with an anti-human retinol-binding protein (RBP) serum were used to study holo-RBP and apo-RBP in human plasma. Three observations were made the technique allowed for the first time to directly and quantitatively analyse holo- and apo-RBP. Holo-RBP represented 97.86 +/- 0.78% and apo-RBP 1.94 +/- 0.73% of the total RBP. All-trans-retinoic acid (RA) was found to bind to apo-RBP and to significantly modify the tertiary structure of the protein; this raises the question of RBP involvement in the transport of RA. reconstitution of holo-RBP using apo-RBP from delipidized serum was achieved only after its incubation with natural all-trans-retinoids such as retinol, 3-dehydroretinol and retinoic acid but not with synthetic analogs of retinoic acid (13-cis-retinoic acid, TMMP, 13-cis-TMMP, TTNPB). It appears that RBP has a structure specificity for natural retinoids.     

Dynamic changes in morphology,gene expression and microbiome in the jejunum of compensatory‐growth rats induced by protein restriction

We previously reported that protein‐restricted rats experienced compensatory growth when they were switched to a normal protein diet (NPD). This study aimed to investigate the changes in gene expression and microbiome in the jejunum of compensatory‐growth rats. Weaned Sprague‐Dawley rats were assigned to an N group, an LN group and an L group. The rats in the L and N groups were fed a low protein diet (LPD) and the NPD respectively. The rats in the LN group were fed with the LPD for 2 weeks, followed by the NPD. The experiment lasted 70 days, and the rats were sacrificed for sampling on days 14, 28 and 70 to determine the jejunal morphology, microbiome and gene expression related to digestive, absorptive and barrier function. The results showed that, although rats in the LN group had temporarily impaired morphology and gene expression in the jejunum on day 14 in response to the LPD, they had improved jejunal morphology and gene expression related to jejunal function on day 28 compared to rats in the N group. This improvement might promote compensatory growth of rats. However, lower expression of genes related to nutrient absorption and undifferentiated villous height (VH) were observed in the jejunum of rats in the LN group on day 70. In contrast, rats in the L group had lower VH on day 28 and day 70, while the expression of absorptive genes increased on day 28 compared to rats in the N group. Additionally, dramatic microbial changes in the jejunum of compensatory‐growth rats were observed, principally for Lactobacillus, Streptococcus, Corynebacterium and Staphylococcus. Moreover, the abundance of Lactobacillus, Streptococcus, Corynebacterium and Staphylococcus significantly correlated with gene expression in the jejunum as revealed by the correlation analysis.