Conformational Changes of the Sweet Protein Monellin as Measured by Fluorescence Emission

Brand, Joseph G.; Çağan, Robert H.; Bayley, Douglas L.

doi:10.3181/00379727-179-42066

Cited by 5 publications

(4 citation statements)

References 6 publications

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“…1 Lower for an excitation wavelength of 295 nm. The fluorescence of the protein solution has its maximum at 342 nm, in agreement with previous reports (19,20). The fluorescence spectrum is practically unaltered for excitation wavelengths between 290 and 300 nm.…”

Section: Resultssupporting

confidence: 91%

Hydration at the surface of the protein Monellin: Dynamics with femtosecond resolution

Peón

Pal²,

Zewail³

2002

Proc. Natl. Acad. Sci. U.S.A.

149

183

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We have studied the femtosecond hydration dynamics of Monellin, a protein with a single tryptophan residue at its surface. Tryptophan was selectively used as a probe of the dynamics, and through monitoring of its fluorescence Stokes shift with time we obtained the hydration correlation function, which decays due to rotational and translational motions of water at the protein surface and in bulk. The decay exhibits a ''bimodal'' behavior with time constants of 1.3 and 16 ps, mirroring relaxation of the free͞quasifree water molecules and surface-bound water layer (minimum binding energy of 1-2 kcal͞mol). The observed slow decay of 16 ps for tryptophan in the native protein differs by more than an order of magnitude from that of bulk water because of the dynamical exchange in the layer. To examine the effect of unfolding, we also studied hydration dynamics when Monellin was denatured in a 6 M guanidine hydrochloride solution and obtained a totally different behavior: 3.5 and 56 ps. Comparing with the results of experiments on free tryptophan in the same concentration of the denaturing solution, we conclude that the fast component of 3.5 ps comes from bulk-type solvation in the 6 M guanidine hydrochloride. However, the absence of the 16-ps decay and appearance of the 56-ps component reflects a more ''rigid solvation,'' which is likely to involve the motions of the protein backbone in the random-coiled state. With the help of polymer theory, this time scale is reproduced in agreement with experimental observations. W ater at the surface of a protein defines a molecular layer that has been termed ''biological water'' and exhibits unique characteristics. The structure and dynamics of such layers, which are determined by the hydrophobic and hydrophilic interactions with the residues exposed to the water in the folded state, are important for the stability of proteins as well as for the mechanisms of protein-protein and protein-ligand association (1). For example, the energetics and dynamics of water desolvation have been suggested to be a determining factor in the process of protein-ligand recognition (2). In molecular dynamics simulations, water molecules have been found to mediate or bridge the interaction between DNA and proteins through their hydrogen bonding (3).In a recent communication (4), we described how the ultrafast Stokes shift of the fluorescence from a single tryptophan amino acid (Trp) at the surface of a protein can be used to probe the dynamics of the biological water layer. Briefly, we use a femtosecond UV pulse to selectively excite the indole chromophore of the Trp residue situated at the protein surface. Although the initial excitation might involve two overlapping transitions for the indole chromophore ( 1 L a and 1 L b ), it has been demonstrated that any electronic mixing that leads to the 1 L a fluorescing state occurs in under 100 fs (5, 6); the 1 L a state of Trp has a significantly larger dipole moment than the ground state. Subsequent to the formation of this dipole by femtosecond excitatio...

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Section: Resultssupporting

confidence: 91%

Hydration at the surface of the protein Monellin: Dynamics with femtosecond resolution

Peón

Pal²,

Zewail³

2002

Proc. Natl. Acad. Sci. U.S.A.

149

183

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“…X‐ray and NMR methods have resolved the structure of monellin with four anti‐parallel β‐sheets and one α‐helix [26,27]. The protein is very stable against acidification of solution, but undergoes heat and chemical denaturation rather easily [28–34]. The denaturation processes are often irreversible [28,29], and the tendency suggests that the system may be useful for studying protein aggregation phenomena.…”

Section: Introductionmentioning

confidence: 99%

Amyloid‐like aggregates of a plant protein: a case of a sweet‐tasting protein, monellin

1999

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We report here a novel case of amyloid-like aggregation of a plant protein. A sweet-tasting protein, monellin, experiences an irreversible heat denaturation at pH 2.5 and 85 degrees C. Addition of 100 mM NaCl couples this process with protein aggregation. The aggregates were structured as regular fibers with approximately 10 nm width and capable of binding to Congo red, similarly to well-known amyloid fibrils. The amyloid-like aggregation process was also successfully monitored with a calorimetric method. This work supports the universality of the amyloid-like aggregation, not restricted to some special categories of protein.

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“…Its two chains, A and B, are held together by interchain hydrophobic interactions, H-bonds, and salt bridges, [47][48][49] and its biological activity (sweetness) is dependent on the integrity of its secondary, tertiary, and quaternary structure. [50][51][52][53][54][55] A sweet single-chain variant of monellin (MNEI or scMN) has been created through genetic fusion, by joining the C-terminal end of chain B to the N-terminal end of chain A with a Gly-Phe linker. Single-chain monellin has been used extensively as a model protein for folding studies.…”

mentioning

confidence: 99%

“…Naturally occurring monellin (dcMN), isolated originally from the berry of an African plant, Dioscoreophyllum cuminisii , is heterodimeric. Its two chains, A and B, are held together by interchain hydrophobic interactions, H-bonds, and salt bridges, − and its biological activity (sweetness) is dependent on the integrity of its secondary, tertiary, and quaternary structure. − A sweet single-chain variant of monellin (MNEI or scMN) has been created through genetic fusion, by joining the C-terminal end of chain B to the N-terminal end of chain A with a Gly-Phe linker. Single-chain monellin has been used extensively as a model protein for folding studies. ,− There have been relatively few studies of the folding of dcMN, , partly because of the heterogeneity observed in the commercially available protein, , but the recent availability of a good bacterial expression system has made dcMN an attractive model protein for the study of the folding of a heterodimeric protein.…”

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confidence: 99%

Equilibrium Unfolding Studies of Monellin: The Double-Chain Variant Appears To Be More Stable Than the Single-Chain Variant

2011

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To improve our understanding of the contributions of different stabilizing interactions to protein stability, including that of residual structure in the unfolded state, the small sweet protein monellin has been studied in both its two variant forms, the naturally occurring double-chain variant (dcMN) and the artificially created single-chain variant (scMN). Equilibrium guanidine hydrochloride-induced unfolding studies at pH 7 show that the standard free energy of unfolding, ΔG°(U), of dcMN to unfolded chains A and B and its dependence on guanidine hydrochloride (GdnHCl) concentration are both independent of protein concentration, while the midpoint of unfolding has an exponential dependence on protein concentration. Hence, the unfolding of dcMN like that of scMN can be described as two-state unfolding. The free energy of dissociation, ΔG°(d), of the two free chains, A and B, from dcMN, as measured by equilibrium binding studies, is significantly lower than ΔG°(U), apparently because of the presence of residual structure in free chain B. The value of ΔG°(U), at the standard concentration of 1 M, is found to be ∼5.5 kcal mol(-1) higher for dcMN than for scMN in the range from pH 4 to 9, over which unfolding appears to be two-state. Hence, dcMN appears to be more stable than scMN. It seems that unfolded scMN is stabilized by residual structure that is absent in unfolded dcMN and/or that native scMN is destabilized by strain that is relieved in native dcMN. The value of ΔG°(U) for both protein variants decreases with an increase in pH from 4 to 9, apparently because of the thermodynamic coupling of unfolding to the protonation of a buried carboxylate side chain whose pK(a) shifts from 4.5 in the unfolded state to 9 in the native state. Finally, it is shown that although the thermodynamic stabilities of dcMN and scMN are very different, their kinetic stabilities with respect to unfolding in GdnHCl are very similar.

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Conformational Changes of the Sweet Protein Monellin as Measured by Fluorescence Emission

Cited by 5 publications

References 6 publications

Hydration at the surface of the protein Monellin: Dynamics with femtosecond resolution

Hydration at the surface of the protein Monellin: Dynamics with femtosecond resolution

Amyloid‐like aggregates of a plant protein: a case of a sweet‐tasting protein, monellin

Equilibrium Unfolding Studies of Monellin: The Double-Chain Variant Appears To Be More Stable Than the Single-Chain Variant

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