pH-induced Structural Changes Research Articles

PH-Dependent Conformational Switch Impacts Stability of the PsbS Dimer.

The photosystem II PsbS protein triggers the photoprotective mechanism of plants by sensing the acidification of the thylakoid lumen. Despite the mechanism of action of PsbS would require a pH-dependent monomerization of the dimeric form, a clear connection between the pH-induced structural changes and the dimer stability is missing. Here, by applying constant pH coarse-grained and all-atom molecular dynamics simulations, we investigate the pH-dependent structural response of the PsbS dimer. We find that the pH variation leads to structural changes in the lumen-exposed helices, located at the dimeric interface, providing an effective switch between PsbS inactive and active form. Moreover, the monomerization free energies reveal that in the neutral pH conformation, where the network of H-bond interactions at the dimeric interface is destroyed, the protein-protein interaction is weaker. Our results show how the pH-dependent conformations of PsbS affect their dimerization propensity, which is at the basis of the photoprotective mechanism.

Elucidation of pH-Induced Protein Structural Changes: A Combined 2D IR and Computational Approach.

The acid-base behavior of amino acids plays critical roles in several biochemical processes. Depending on the interactions with the protein environment, the pKa values of these amino acids shift from their respective solution values. As the side chains interact with the polypeptide backbone, a pH-induced change in the protonation state of aspartic and glutamic acids might significantly influence the structure and stability of a protein. In this work, we have combined two-dimensional infrared spectroscopy and molecular dynamics simulations to elucidate the pH-induced structural changes in an antimicrobial enzyme, lysozyme, over a wide range of pH. Simultaneous measurements of the carbonyl signals arising from the backbone and the acidic side chains provide detailed information about the pH dependence of the local and global structural features. An excellent agreement between the experimental and the computational results allowed us to obtain a residue-specific molecular understanding. Although lysozyme retains the helical structure for the entire pH range, one distinct loop region (residues 65-75) undergoes local structural deformation at low pH. Interestingly, combining our experiments and simulations, we have identified the aspartic acid residues in lysozyme, which are influenced the most/least by pH modulation.

Scanning pH-metry for Observing Reversibility in Protein Folding.

One of the main factors affecting protein structure in solution is pH. Traditionally, to study pH-dependent conformational changes in proteins, the concentration of the H+ ions is adjusted manually, complicating real-time analyses, hampering dynamic pH regulation, and consequently leading to a limited number of tested pH levels. Here, we present a programmable device, a scanning pH-meter, that can automatically generate different types of pH ramps and waveforms in a solution. A feedback loop algorithm calculates the required flow rates of the acid/base titrants, allowing one, for example, to generate periodic pH sine waveforms to study the reversibility of protein folding by fluorescence spectroscopy. Interestingly, for some proteins, the fluorescence intensity profiles recorded in such a periodically oscillating pH environment display hysteretic behavior indicating an asymmetry in the sequence of the protein unfolding/refolding events, which can most likely be attributed to their distinct kinetics. Another useful application of the scanning pH-meter concerns coupling it with an electrospray ionization mass spectrometer to observe pH-induced structural changes in proteins as revealed by their varying charge-state distributions. We anticipate a broad range of applications of the scanning pH-meter developed here, including protein folding studies, determination of the optimum pH for achieving maximum fluorescence intensity, and characterization of fluorescent dyes and other synthetic materials.

Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH

The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.

Investigation of a KcsA Cytoplasmic pH Gate in Lipoprotein Nanodiscs.

The bacterial potassium channel KcsA is gated by pH, opening for conduction under acidic conditions. Molecular determinants responsible for this effect have been identified at the extracellular selectivity filter, at the membrane-cytoplasm interface (TM2 gate), and in the cytoplasmic C-terminal domain (CTD), an amphiphilic four-helix bundle mediated by hydrophobic and electrostatic interactions. Here we have employed NMR and EPR to provide a structural view of the pH-induced open-to-closed CTD transition. KcsA was embedded in lipoprotein nanodiscs (LPNs), selectively methyl-protonated at Leu/Val residues to allow observation of both states by NMR, and spin-labeled for the purposes of EPR studies. We observed a pHinduced structural change between an associated structured CTD at neutral pH and a dissociated flexible CTD at acidic pH, with a transition in the 5.0-5.5 range, consistent with a stabilization of the CTD by channel architecture. A double mutant constitutively open at the TM2 gate exhibited reduced stability of associated CTD, as indicated by weaker spin-spin interactions, a shift to higher transition pH values, and a tenfold reduction in the population of the associated "closed" channels. We extended these findings for isolated CTD-derived peptides to full-length KcsA and have established a contribution of the CTD to KcsA pH-controlled gating, which exhibits a strong correlation with the state of the proximal TM2 gate.

Dual hepatocyte-targeting fluorescent probe with high sensitivity to tumorous pH: Precise detection of hepatocellular carcinoma cells

A new dual hepatocyte-targeting fluorescent probe HPL-1, which can precisely distinguish tumorous pH from physiological pH, was developed. The OFF-ON switch of HPL-1 can be triggered via pH-induced structural change of the lactam group of the rhodamine moiety from closed-ring to open-ring. Our results showed that the phosphate group of HPL-1 is beneficial to its accumulation in liver cells, and combination of the phosphate and galactose units could synergistically increase the hepatocyte-targeting capacity. HPL-1 could selectively distinguish hepatoma cells from other tissue cells, and precisely distinguish cancerous liver cells from normal liver cells. Compared with other reported probes, HPL-1 not only enable a simple and convenient detection method, but also has good hepatocyte-targeting capacity and precise recognition capacity of tumors under weak acid micro-environment, which opens new avenues for precise diagnosis and treatment of hepatocellular carcinoma.

Folding of poly-amino acids and intrinsically disordered proteins in overcrowded milieu induced by pH change

pH-induced structural changes of the synthetic homopolypeptides poly-E, poly-K, poly-R, and intrinsically disordered proteins (IDPs) prothymosin α (ProTα) and linker histone H1, in concentrated PEG solutions simulating macromolecular crowding conditions within the membrane-less organelles, were characterized. The conformational transitions of the studied poly-amino acids in the concentrated PEG solutions depend on the polymerization degree of these homopolypeptides, the size of their side chains, the charge distribution of the side chains, and the crowding agent concentration. The results obtained for poly-amino acids are valid for IDPs having a significant total charge. The overcrowded conditions promote a significant increase in the cooperativity of the pH-induced coil–α-helix transition of ProTα and provoke histone H1 aggregation. The most favorable conditions for the pH-induced structural transitions in concentrated PEG solutions are realized when the charged residues are grouped in blocks, and when the distance between the end of the side group carrying charge and the backbone is small. Therefore, the block-wise distribution of charged residues within the IDPs not only plays an important role in the liquid–liquid phase transitions, but may also define the expressivity of structural transitions of these proteins in the overcrowded conditions of the membrane-less organelles.

PH-induced structural changes of apo-lactoferrin and implications for its function: a molecular dynamics simulation study

ABSTRACTThe minor protein in milk, lactoferrin (Lf), is known for a variety of biological functions, and has been investigated as a protective encapsulant for probiotic bacteria in health-promoting food products. Lf is likely to be exposed to extreme pH conditions which are known to have disruptive influences on its functionality. The molecular mechanisms underlying these pH-dependent changes are not well-understood. To explore the potential of Lf as an encapsulant, molecular dynamics (MD) simulations were applied to study its conformational changes under extreme acidic (pH 1.0) or basic (pH 14.0) conditions, relative to neutral pH. Simulations indicate that the structure of apo-Lf is relatively stable at neutral pH, while acidic and basic pH result in substantially greater flexibility, partly induced by the loss of contacts between the N- and C-terminal lobes, causing them to undergo extensive relative bending and twisting motions. Basic pH causes greater structural disruption compared to acidic exposure. The latter has greater influence on the N-terminus, with increased fluctuations and disruptions of inter-residue contacts compared to those at neutral pH; while basic pH was found to more prominently disrupt contacts at the C-terminus. These results help elucidate possible functional consequences on Lf of exposure to extreme pH conditions.

The acidic pH-induced structural changes in apo-CP43 by spectral methodologies and molecular dynamics simulations

CP43 is closely associated with the photosystem II and exists the plant thylakoid membranes. The acidic pH-induced structural changes had been investigated by fluorescence spectrum, ANS spectrum, RLS spectrum, energy transfer experiment, acrylamide fluorescence quenching assay and MD simulation. The fluorescence spectrum indicated that the structural changes in acidic pH-induced process were a four-state model, which was nature state (N), partial unfolding state (PU), refolding state (R), and molten-globule state (M), respectively. Analysis of ANS spectrum illustrated that inner hydrophobic core exposed partially to surface below pH 2.0 and inferred also that the molten-globule state existed. The RLS spectrum showed the aggregation of apo-CP43 around the pI (pH 4.5–4.0). The alterations of apo-CP43 secondary structure with different acidic treatments were confirmed by FTIR spectrum. The energy transfer experiment and quenching research demonstrated structural change at pH 4.0 was loosest. The RMSF suggested two terminals played an important function in acidic denaturation process. The distance of two terminals shown slight difference in acidic pH-induced process during the unfolding process, both N-terminal and C-terminal occupied the dominant role. However, the N-terminal accounted for the main part in the refolding process. All kinds of SASA values corresponded to spectral results. The tertiary and secondary structure by MD simulation indicated that the part transmembrane α-helix was destroyed at low pH.

Influence of pH on the Structure and Function of Kiwi Pectin Methylesterase Inhibitor.

Pectin methylesterase is a pectin modifying enzyme that plays a key role in plant physiology. It is also an important quality-related enzyme in plant-based food products. The pectin methylesterase inhibitor (PMEI) from kiwifruit inhibits this enzyme activity and is widely used as an efficient tool for research purposes and also recommended in the context of fruit and vegetable processing. Using several methodologies of protein biochemistry, including circular dichroism and fluorescence spectroscopy, chemical modifications, direct protein-sequencing, enzyme activity, and bioinformatics analysis of the crystal structure, this study demonstrates that conformational changes occur in kiwi PMEI by the pH rising over 6.0 bringing about structure loosening, exposure, and cleavage of a natively buried disulfide bond, unfolding and aggregation, ultimately determining the loss of ability of kiwi PMEI to bind and inhibit PME. pH-induced structural changes are prevented when PMEI is already engaged in complex or is in a solution of high ionic strength.

Initial Droplet Size Impacts pH-Induced Structural Changes in Phase-Separated Polymer Dispersions.

The effect of pH change on the morphology of whey protein isolate (WPI)-pectin dispersions obtained from phase-separated systems after mild shear was studied. The purpose of this study was to examine the impact of mixing speed on the initial particle size of biopolymer complexes and their structure morphology after sequentially changing the pH. Therefore, solutions of WPI and pectin were combined at pH 6.1, allowed to phase separate and were then mildly homogenized at 50, 100, and 150 rpm, respectively, to form a dispersion containing differently sized WPI droplets in a surrounding pectin-rich phase. Each dispersion was then subjected to a pH change, such as 6.1 to 5.2 and 3.2, by slowly adding hydrochloric acid. The systems morphology, size, appearance, rheology, and storage stability was then characterized by optical microscopy, static light scattering, visual inspections, and steady shear rheometry to gain insights into the structural rearrangements. Results indicated substantial changes in the structure of the dispersion when the pH was changed. Formation of core-shell structures from the WPI droplets was observed at an intermediate pH. There, initial droplet size was found to affect structures formed, that is, core-shell type particles would only form if droplets were large (>1.5 μm) prior to pH change. Insights gained may be of importance to food manufacturers intending to create new structures from mixtures of proteins and carbohydrates.

Two-dimensional (2D) infrared correlation study of the structural characterization of a surface immobilized polypeptide film stimulated by pH

The pH-induced structural changes to surface immobilized poly (l-glutamic acid) (PLGA) films were examined by Fourier transform infrared (FTIR) spectroscopy and two-dimensional (2D) correlation analysis. Significant spectral changes were observed in the FTIR spectra of the surface immobilized PLGA film between pH 6 and 7. The 2D correlation spectra constructed from the pH-dependent FTIR spectra of the surface immobilized PLGA films revealed the spectral changes induced by the alternations of the protonation state of the carboxylic acid group in the PLGA side chain. When the pH was increased from 6 to 8, weak spectral changes in the secondary structure of the PLGA main chain were induced by deprotonation of the carboxylic acid side group.

Role of pH-induced structural change in protein aggregation in foam fractionation of bovine serum albumin

For reducing protein aggregation in foam fractionation, the role of pH-induced structural change in the interface-induced protein aggregation was analyzed using bovine serum albumin (BSA) as a model protein. The results show that the decrease in pH from 7.0 to 3.0 gradually unfolded the BSA structure to increase the molecular size and the relative content of β-sheet and thus reduced the stability of BSA in the aqueous solution. At the isoelectric point (pH 4.7), BSA suffered the lowest level in protein aggregation induced by the gas–liquid interface. In the pH range from 7.0 to 4.7, most BSA aggregates were formed in the defoaming process while in the pH range from 4.7 to 3.0, the BSA aggregates were formed at the gas–liquid interface due to the unfolded BSA structure and they further aggregated to form insoluble ones in the desorption process.

A Dynamic Model of pH-Induced Protein G'e Higher Order Structure Changes derived from Mass Spectrometric Analyses.

To obtain insight into pH change-driven molecular dynamics, we studied the higher order structure changes of protein G'e at the molecular and amino acid residue levels in solution by using nanoESI- and IM-mass spectrometry, CD spectroscopy, and protein chemical modification reactions (protein footprinting). We found a dramatic change of the overall tertiary structure of protein G'e when the pH was changed from neutral to acidic, whereas its secondary structure features remained nearly invariable. Limited proteolysis and surface-topology mapping of protein G'e by fast photochemical oxidation of proteins (FPOP) under neutral and acidic conditions reveal areas where higher order conformational changes occur on the amino-acid residue level. Under neutral solution conditions, lower oxidation occurs for residues of the first linker region, whereas greater oxidative modifications occur for amino-acid residues of the IgG-binding domains I and II. We propose a dynamic model of pH-induced structural changes in which protein G'e at neutral pH adopts an overall tight conformation with all four domains packed in a firm assembly, whereas at acidic pH, the three IgG-binding domains form an elongated alignment, and the N-terminal, His-tag-carrying domain unfolds. At the same time the individual IgG-binding domains themselves seem to adopt a more compacted fold. As the secondary structure features are nearly unchanged at either pH, interchange between both conformations is highly reversible, explaining the high reconditioning power of protein G'e-based affinity chromatography columns.

Structural and functional analysis of botulinum neurotoxin subunits for pH-dependent membrane channel formation and translocation

The structure–function relationship of Botulinum Neurotoxin (BoNT) proteins is greatly influenced by pH. While the low pH of endosome favors membrane interaction of the heavy chain (HC) for the formation of a membrane channel and translocation of the light chain (LC), the catalytic activity of the LC requires a neutral pH for cleavage of the soluble NSF attachment protein receptor (SNARE) complex in the cytosol. In this study, we monitored secondary structural characteristics of LC, HC and holotoxin at individual pHs 4.5 and 7.2 and at the transition pH4.5 to 7.2 to identify the structural signatures underlying their function. The HC showed higher thermal stability at pH4.5 with a melting temperature (Tm) of 60.4°C. The structural analysis of HC in the presence of liposomes showed no difference in ellipticity with that of HC at pH7.2 at 208 and 222nm but a 25.2% decrease in ellipticity at 208nm at acidic pH, indicating low pH-induced structural changes that might facilitate interaction with the membrane. Further, HC showed 18% release of K+ ions from liposomes at pH4.5 as against 6% at neutral pH, reinforcing its role in membrane channel formation. LC on the other hand, showed maximum ellipticity at pH7.2, a condition that is relevant to its endopeptidase activity in the cytosol of the neurons. Also, the similarity in the structures at pH7.2 and transition pH4.5 to 7.2 suggested that the flexibility acquired by the protein at low pH was reversible upon exposure to neutral pH for cleavage of SNARE proteins.

PH-induced structural change of a multi-tryptophan protein MPT63 with immunoglobulin-like fold: identification of perturbed tryptophan residue/residues

The structural change of M. tuberculosis MPT63, which is predominantly a β-sheet protein having an immunoglobulin like fold, has been investigated in the pH range 7.5–1.5 using various biophysical techniques along with low-temperature phosphorescence (LTP) spectroscopy. MPT63 contains four Tryptophan (Trp) residues at 26, 48, 82, and 129. Although circular dichroism, steady-state and time-resolved fluorescence, time-resolved anisotropy, 1-aniline-8-naphthalene sulfonic (ANS) acid binding, and analytical ultracentrifuge depict more open largely unfolded structure of MPT63 at pH 1.5 and also more accessible nature of Trp residues to neutral quencher at pH 1.5, it is, however, not possible to assign the specific Trp residue/residues being perturbed. This problem has been resolved using LTP of MPT63, which shows optically resolved four distinct (0, 0) bands corresponding to four Trp residues in the pH range 7.5–3.0. LTP at pH 1.5 clearly reveals that the solvent-exposed Trp 82 and the almost buried Trp 129 are specifically affected compared with Trp 48 and Trp 26. Lys 8 and Lys 27 are predicted to affect Trp 129. Tyrosine residues are found to be silent even at pH 1.5. This type of specific perturbation in a multi-Trp protein has not been addressed before. LTP further indicates that partially exposed Trp 48 is preferentially quenched by acrylamide compared with other Trp residues at both pH 7.5 and 1.5. The solvent-exposed Trp 82 is surprisingly found to be not quenched by acrylamide at pH 7.5. All the results are obtained using micromolar concentration of protein and without using any Trp-substituted mutant.

PH-induced structural changes of surface immobilized poly(l-lysine) by two-dimensional (2D) infrared correlation study

Abstract This paper reports the pH-induced structural changes in the surface immobilized poly( l -lysine) (PLL) film. Two-dimensional (2D) correlation analysis was applied to the Fourier transform infrared (FTIR) spectra of the surface-immobilized PLL film to examine the spectral changes induced by the alternations of the protonation state of the amino group in the side chain. Significant spectral changes in the FTIR spectra of the PLL film were observed between pH 7 and 8. The decrease in the protonation state of the amino group in the side chain induced spectral changes in the amino group as well as conformational changes in the alkyl group in the side chain. From pH 1–8, the spectral changes in the amino and alkyl groups in the side chain occurred before those of the amide group in the main chain of the surface immobilized PLL film.

The cellular receptor for enterovirus 71.

The cellular receptor for enterovirus 71.

PH-induced structural changes of ovalbumin studied by 2D correlation IR spectroscopy

The secondary structural changes of pH-induced ovalbumin during the transition from native state into intermediate state were studied with the use of 2D correlation spectroscopy and principal component analysis. 2D correlation spectra constructed from the pH-dependent IR spectra of ovalbumin solution revealed the following scenario of the intensity changes with pH decrease. When pH decreased from 5.5 and 3.6 intensity of components attributed to the β-turns, the α-helical elements, and native β-sheets increased. It was caused by protonation induced changes in environment of these elements. When the protonation of the acidic groups were finalized the system adopted the intermediate structure. It was accompanied by weak structural changes that mainly included the β-turns and the α-helices. In extreme acidic conditions at pH below pH 2 the intermediate structure was no longer stable and oligomers rich in the β-sheet structure were formed.

PH-Induced conformational changes of BSA in fluorescent AuNCs@BSA and its effects on NCs emission

We investigated the pH-induced fluorescence changes of BSA-protected gold nanoclusters, Au16NCs@BSA, and the corresponding conformational changes of ligand protein by fluorescence, circular dichrosim (CD) and IR spectral measurements. The studies presented here demonstrated that BSA in AuNCs@BSA underwent identifiable conformational changes on both the secondary and the tertiary structure levels. The results of CD and IR interpreted the significant change of second structures at extreme acidity and alkaline, where more unordered structures were gained. Of note was that the extreme alkaline (pH=11.43) induced the changes from exposed to buried α-helices, which was different from the pH-induced structural changes of BSA. In addition, the large fluorescence intensity gap of tryptophan between AuNCs@BSA and native BSA indicated efficient energy transfer took place between BSA and AuNCs, implying that the gold core resided near tryptophan in BSA.