Single-chain Monellin Research Articles

The Effect of Nanoparticles on Amyloid Aggregation Depends on the Protein Stability and Intrinsic Aggregation Rate

Nanoparticles interfere with protein amyloid formation. Catalysis of the process may occur due to increased local protein concentration and nucleation on the nanoparticle surface, whereas tight binding or a large particle/protein surface area may lead to inhibition of protein aggregation. Here we show a clear correlation between the intrinsic protein stability and the nanoparticle effect on the aggregation rate. The results were reached for a series of five mutants of single-chain monellin differing in intrinsic stability toward denaturation, for which a correlation between protein stability and aggregation propensity has been previously documented by Szczepankiewicz et al. [Mol. Biosyst.20107 (2), 521–532]. The aggregation process was monitored by thioflavin T fluorescence in the absence and presence of copolymeric nanoparticles with different hydrophobic characters. For mutants with a high intrinsic stability and low intrinsic aggregation rate, we find that amyloid fibril formation is accelerated by nanoparticles. For mutants with a low intrinsic stability and high intrinsic aggregation rate, we find the opposite—a retardation of amyloid fibril formation by nanoparticles. Moreover, both catalytic and inhibitory effects are most pronounced with the least hydrophobic nanoparticles, which have a larger surface accessibility of hydrogen-bonding groups in the polymer backbone.

Identification of Multiple Folding Pathways of Monellin Using Pulsed Thiol Labeling and Mass Spectrometry

Protein folding reactions often display multiexponential kinetics of changes in intrinsic optical signals, as a manifestation of heterogeneity, either on one folding pathway or on multiple folding pathways. Delineating the origin of this heterogeneity is difficult because different coexisting structural forms of a protein cannot be easily distinguished by optical probes. In this study, the complex folding reaction of single-chain monellin has been investigated using a pulsed thiol labeling (SX) methodology in conjunction with mass spectrometry, which measures the kinetics of burial of a cysteine side chain thiol during folding. Because it can directly distinguish between unfolded and folded molecules and can measure the disappearance of the former during folding, the pulsed SX methodology is an ideal method for investigating whether multiple pathways are operative during folding. The kinetics of burial of the C42 thiol of monellin was observed to follow biexponential kinetics. To determine whether this was because the fast phase leads to the partial protection of the thiol group in all the molecules or to complete protection in only a fraction of the molecules, the duration and intensity of the labeling pulse were varied. The observation that the extent of labeling did not vary with the duration of the pulse cannot be explained by a simple sequential folding mechanism. Two parallel folding pathways are shown to be operative, with one leading to the formation of thiol-protective structure more rapidly than the other.

Reduced Sweetness of a Monellin (MNEI) Mutant Results from Increased Protein Flexibility and Disruption of a Distant Poly-(L-Proline) II Helix

Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding.

Interactions in the native state of monellin, which play a protective role against aggregation

A series of recent studies have provided initial evidence about the role of specific intra-molecular interactions in maintaining proteins in their soluble state and in protecting them from aggregation. Here we show that the amino acid sequence of the protein monellin contains two aggregation-prone regions that are prevented from initiating aggregation by multiple non-covalent interactions that favor their burial within the folded state of the protein. By investigating the behavior of single-chain monellin and a series of five of its mutational variants using a variety of biochemical, biophysical and computational techniques, we found that weakening of the non-covalent interaction that stabilizes the native state of the protein leads to an enhanced aggregation propensity. The lag time for fibrillation was found to correlate with the apparent midpoint of thermal denaturation for the series of mutational variants, thus showing that a reduced thermal stability is associated with an increased aggregation tendency. We rationalize these findings by showing that the increase in the aggregation propensity upon mutation can be predicted in a quantitative manner through the increase in the exposure to solvent of the amyloidogenic regions of the sequence caused by the destabilization of the native state. Our findings, which are further discussed in terms of the structure of monellin and the perturbation by the amino acid substitutions of the contact surface between the two subdomains that compose the folded state of monellin, provide a detailed description of the specific intra-molecular interactions that prevent aggregation by stabilizing the native state of a protein.

Expression and secretion of a single-chain sweet protein, monellin, in Saccharomyces cerevisiae by an α-factor signal peptide

The sweet protein monellin gene was expressed in Saccharomyces cerevisiae under the control of the GAL1 promoter and α-factor signal peptide sequence of S. cerevisiae. The gene, which was obtained through mutation of the synthesized single-chain monellin gene, was cloned into an E. coli-yeast shuttle vector pYES2.0 which carries the galactose-inducible promoter GAL1. Then the α-factor signal peptide of S. cerevisiae was linked also, resulting in the secreting expression vector pYESMTA. The recombinant plasmid was subsequently transformed into strain S. cerevisiae INVsc1. The peptide efficiently directed the secretion of monellin from the recombinant yeast cell. A maximum yield of active monellin was 0.41 g l(-1) of the supernatant from INVsc1 harboring pYESMTA.

Heterologous expression, purification and characterization of heterodimeric monellin

Monellin is an intensely sweet-tasting protein present in the berry of Dioscoreophyllum cumminsii. Naturally occurring monellin (double chain monellin) is a heterodimer of two subunits commonly referred to as chain A and chain B. Monellin is a good model system for structural and dynamic studies of proteins. Single chain monellin has been generated by covalently linking the two subunits of naturally occurring double chain monellin, and has been used extensively for folding and unfolding studies, as well as for protein aggregation studies. There are, however, relatively few reports on such studies with double chain monellin. The primary difficulty associated with studies using double chain monellin appears to be the lack of a standard purification method. Here, a simple method for the purification of double chain monellin is presented. The genes encoding the two chains of monellin were cloned into a modified pETDUET vector under separate T7 promoters. The expression vector containing the genes of the two chains was expressed in E. coli BL21 Star (DE3). The expressed protein was purified using two steps of chromatography, ion exchange chromatography and gel filtration chromatography. This expression system consistently produced 40mg of pure double chain monellin per litre of E. coli culture, in the correctly folded native state. The purity of the protein was confirmed by mass spectrometry and SDS–PAGE analysis. The purified protein was characterized using different spectroscopic methods, and the spectra obtained were in good agreement with the published spectra of naturally occurring double chain monellin.

Aggregation Mechanisms of Cystatins: A Comparative Study of Monellin and Oryzacystatin

Identification of diseases caused by protein misfolding has increased interest in the way proteins adopt non-native conformations and form aggregates. In this study we address the question of how proteins sharing the same fold respond to destabilizing environmental conditions. We have studied the behavior of two members of the cystatin superfamily, MNEI, a single chain monellin, and oryzacystatin_I, a plant cystatin. Despite the close similarity of their three-dimensional architecture, these two proteins aggregate in a different way: MNEI gives rise to amyloid aggregation whereas oryzacystatin_I yields amorphous aggregates. Mutants of oryzacystatin_I, designed to make it more similar to MNEI, generally behave like the parent protein, but a construct devoid of the disordered N- and C-terminal sequences does show a tendency to form amyloid fibers. Our results suggest that precise sequence details may be more important than the three-dimensional architecture in determining the type of aggregate formed. Oryzacystatin_I appears to be a very promising model system for further studies of protein aggregation.

Direct evidence for a dry molten globule intermediate during the unfolding of a small protein

Little is known about how proteins begin to unfold. In particular, how and when water molecules penetrate into the protein interior during unfolding, thereby enabling the dissolution of specific structure, is poorly understood. The hypothesis that the native state expands initially into a dry molten globule, in which tight packing interactions are broken, but whose hydrophobic core has not expanded sufficiently to be able to absorb water molecules, has very little experimental support. Here, we report our analysis of the earliest observable events during the unfolding of single chain monellin (MNEI), a small plant protein. Far- and near-UV circular dichroism measurements of GdnHCl-induced unfolding indicate that a molten globule intermediate forms initially, before the major slow unfolding reaction commences. Steady-state fluorescence resonance energy transfer measurements show that the C-terminal end of the single helix of MNEI initially moves rapidly away from the single tryptophan residue that is close to the N-terminal end of the helix. The average end-to-end distance of the protein also expands during unfolding to the molten globule intermediate. At this time, water has yet to penetrate the protein core, according to the evidence from intrinsic tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid fluorescence-monitored kinetic unfolding measurements. Our results therefore provide direct evidence for a dry molten globule intermediate at the initial stage of unfolding. Our results further suggest that the structural transition between the native and dry molten globule states could be an all-or-none transition, whereas further swelling of the globule appears to occur gradually.

GroEL Can Unfold Late Intermediates Populated on the Folding Pathways of Monellin

The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50–60% of monellin molecules fold completely. The remaining 40–50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.

Role of protein surface charge in monellin sweetness

A small number of proteins have the unusual property of tasting intensely sweet. Despite many studies aimed at identifying their sweet taste determinants, the molecular basis of protein sweetness is not fully understood. Recent mutational studies of monellin have implicated positively charged residues in sweetness. In the present work, the effect of overall net charge was investigated using the complementary approach of negative charge alterations. Multiple substitutions of Asp/Asn and Glu/Gln residues radically altered the surface charge of single-chain monellin by removing six negative charges or adding four negative charges. Biophysical characterization using circular dichroism, fluorescence, and two-dimensional NMR demonstrates that the native fold of monellin is preserved in the variant proteins under physiological solution conditions although their stability toward chemical denaturation is altered. A human taste test was employed to determine the sweetness detection threshold of the variants. Removal of negative charges preserves monellin sweetness, whereas added negative charge has a large negative impact on sweetness. Meta-analysis of published charge variants of monellin and other sweet proteins reveals a general trend toward increasing sweetness with increasing positive net charge. Structural mapping of monellin variants identifies a hydrophobic surface predicted to face the receptor where introduced positive or negative charge reduces sweetness, and a polar surface where charges modulate long-range electrostatic complementarity.

Dehydration of main-chain amides in the final folding step of single-chain monellin revealed by time-resolved infrared spectroscopy

Kinetic IR spectroscopy was used to reveal beta-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded beta-sheet and an alpha-helix. The time-resolved IR spectra between 100 mus and 10 s were analyzed based on two consecutive intermediates, I(1) and I(2), appearing within 100 mus and with a time constant of approximately 100 ms, respectively. The initial unfolded state showed broad amide I' corresponded to a fluctuating conformation. In contrast, I(1) possessed a feature at 1,636 cm(-1) for solvated helix and weak features assignable to turns, demonstrating the rapid formation of helix and turns. I(2) possessed a line for solvated helix at 1,637 cm(-1) and major and minor lines for beta-sheet at 1,625 and 1,680 cm(-1), respectively. The splitting of the major and minor lines is smaller than that of the native state, implying an incomplete formation of the beta-sheet. Furthermore, both major and minor lines demonstrated a low-frequency shift compared to those of the native state, which was interpreted to be caused by hydration of the C O group in the beta-sheet. Together with the identification of solvated helix, the core domain of I(2) was interpreted as being hydrated. Finally, slow conversion of the water-penetrated core of I(2) to the dehydrated core of the native state was observed. We propose that both the expulsion of water, hydrogen-bonded to main-chain amides, and the completion of the secondary structure formation contribute to the energetic barrier of the rate-limiting step in SMN folding.

Characterization of the Folding and Unfolding Reactions of Single-Chain Monellin: Evidence for Multiple Intermediates and Competing Pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.

Monellin (MNEI) at 1.15 A resolution.

The X-ray crystal structure of a single-chain monellin protein (MNEI) has been determined at 1.15 A resolution. The model was refined to convergence employing anisotropic displacement parameters and riding H atoms to produce a final model with R(work) and R(free) values of 0.132 and 0.162, respectively. The crystal contains a single MNEI protein in the asymmetric unit and unusually lacks the dimer interface observed in all previous crystal structures of monellin and its single-chain derivatives. The high resolution allowed a more detailed view of MNEI than previously possible, with 38 of the 96 residues modelled with alternative side-chain conformations, including four core residues Thr12, Cys41, Leu62 and Ile75. Four stably bound negative ions were also located, providing new insight into potential electrostatic interactions of MNEI with the largely negatively charged surface of the sweet taste receptor T1R2-T1R3.

Expression and secretion of a single-chain sweet protein monellin in Bacillus subtilis by sacB promoter and signal peptide

The sweet protein monellin gene was expressed in Bacillus subtilis under the control of the Bacillus subtilis sacB promoter and signal peptide sequence. A 294-bp DNA fragment, coding for sweet protein monellin, was ligated into the Escherichia coli/B. subtilis shuttle vector pHPC, producing pHPMS, which was subsequently transformed into B. subtilis QB1098, DB104, and DB403. The peptide efficiently directed the secretion of monellin from the recombinant B. subtilis cells. A maximum yield of monellin of 0.29 g protein l(-1) was obtained from the supernatant of B. subtilis DB403 harboring pHPMS. SDS-PAGE confirmed the purity of the recombinant product.

The Importance of Electrostatic Potential in The Interaction of Sweet Proteins with the Sweet Taste Receptor

In addition to many small molecular mass sweeteners there are in nature a few sweet proteins. The molecular volume of sweet proteins is so different from that of common sweeteners that it was difficult to understand how molecules as large as proteins can activate a receptor designed to host small molecules. We have recently shown that sweet proteins can activate the sweet receptor by a mechanism of interaction, called ‘‘wedge model”, in which proteins fit a large cavity of the receptor with wedge-shaped surfaces of their structures. In order to substantiate this model we have designed, expressed and characterized seven mutants of MNEI, a single chain monellin. Three uncharged residues of the interaction surface, Met42, Tyr63 and Tyr65, were changed either into acidic or basic residues whereas Asp68, a key acidic residue, was changed into a basic one. As a general trend, we observe that an increase of the negative charge is much more detrimental for sweetness than an increase of positive charge. In addition we show that by a careful choice of a residue at the center of the interface between MNEI and receptor, it is possible even to increase the sweetness of MNEI. These results are fully consistent with the wedge model.

Intra- versus Intermolecular Interactions in Monellin: Contribution of Surface Charges to Protein Assembly

The relative significance of weak non-covalent interactions in biological context has been much debated. Here, we have addressed the contribution of Coulombic interactions to protein stability and assembly experimentally. The sweet protein monellin, a non-covalently linked heterodimeric protein, was chosen for this study because of its ability to spontaneously reconstitute from separated fragments. The reconstitution of monellin mutants containing large surface charge perturbations was compared to the thermostability of structurally equivalent single-chain monellin containing the same sets of mutations under varying salt concentrations. The affinity between monellin fragments is found to correlate with the thermostability of single chain monellin, indicating the involvement of the same underlying Coulombic interactions. This confirms that there are no principal differences in the interactions involved in folding and binding. Based on comparison with a previous mutational study involving hydrophobic core residues, the relative contribution of Coulombic interactions to stability and affinity is modest. However, the Coulombic perturbations only affect the association rates of reconstitution in contrast to perturbations involving hydrophobic residues, which affect primarily the dissociation rates. These results indicate that Coulombic interactions are likely to be of main importance for the association of protein assembly, relevant for functions of proteins.

1P112 Multiple time scales in the folding dynamics of single chain monellin revealed by single molecule measurements(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P112 Multiple time scales in the folding dynamics of single chain monellin revealed by single molecule measurements(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

High-level Expression of a Synthetic Gene Encoding a Sweet Protein, Monellin, in Escherichia coli

The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased codons of E. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE.

Specific collapse followed by slow hydrogen-bond formation of β-sheet in the folding of single-chain monellin

Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of single-chain monellin composed of a five-stranded beta-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively. The formation of the tertiary structure was monitored by intrinsic and extrinsic fluorescence. A significant collapse was observed within 300 micros after the pH-jump, leading to the intermediate with a small amount of secondary and tertiary structures but with an overall oblate shape. Subsequently, the stepwise formation of secondary and tertiary structures was detected. The current observation was consistent with the theoretical prediction that a more significant collapse precedes the formation of secondary structures in the folding of beta-sheet proteins than that of helical proteins [Shea, J. E., Onuchic, J. N. & Brooks, C. L., III (2002) Proc. Natl. Acad. Sci. USA 99, 16064-16068]. Furthermore, it was implied that the initial collapse was promoted by the formation of some specific structural elements, such as tight turns, to form the oblate shape.

On the mechanism of chaperone activity of the small heat-shock protein of Methanococcus jannaschii.

The small heat-shock protein (sHSP) from Methanococcus jannaschii (Mj HSP16.5) forms a homomeric complex of 24 subunits and has an overall structure of a multiwindowed hollow sphere with an external diameter of approximately 120 A and an internal diameter of approximately 65 A with six square "windows" of approximately 17 A across and eight triangular windows of approximately 30 A across. This sHSP has been known to protect other proteins from thermal denaturation. Using purified single-chain monellin as a substrate and a series of methods such as protease digestion, antibody binding, and electron microscopy, we show that the substrates bind to Mj HSP16.5 at a high temperature (80 degrees C) on the outside surface of the sphere and are prevented from forming insoluble substrate aggregates in vitro. Circular dichroism studies suggest that a very small, if any, conformational change occurs in sHSP even at 80 degrees C, but substantial conformational changes of the substrate are required for complex formation at 80 degrees C. Furthermore, deletion mutation studies of Mj HSP16.5 suggest that the N-terminal region of the protein has no structural role but may play an important kinetic role in the assembly of the sphere by "preassembly condensation" of multiple monomers before final assembly of the sphere.