Protein Stability in Reverse Micelles

Abstract

The N-terminal SH3 domain of the Drosophila signal transduction protein drk was encapsulated in reverse micelles.B oth the temperature of maximum stability and the melting temperature decreased on encapsulation. Dissecting the temperature-dependent stability into enthalpic and entropic contributions reveals astabilizing enthalpic and adestabilizing entropic contribution. These results do not matcht he expectations of hard-core excluded volume theory,n or can they be wholly explained by interactions between the head groups in the reverse micelle and the test protein. We suggest that geometric constraints imposed by the reverse micelles need to be considered.Understanding the effect of confinement is essential for explaining protein behavior during biosynthesis (ribosome exit tunnel), misfolding (chaperones), turnover (proteasome), and transport across membranes (translocon). Confinement is also important for industrial applications such as enzyme stabilization and drug delivery.[1] Even though confined and crowded environments are intrinsic to biology,t hey are absent from most studies of proteins,which are conducted in simple buffer solutions. [2] Efforts to explain biomolecular interactions in crowded environments have attracted the attention of researchers for decades,b ut the landscape of crowding research has shifted dramatically in the past few years.T he long-accepted explanation for differences in protein chemistry between buffer and the cytoplasm focused on hard-core repulsions, apurely entropic effect.[3] It is now known that repulsions are often diminished or even overwhelmed by nonspecific attractive interactions between the test protein and the macromolecules (and perhaps metabolites) in the cytoplasm and in cosolute solutions in vitro. [4] Confinement is easy to envision. Thet est protein is trapped in ac avity just large enough to contain it and some hydrating water. Aw idely accepted model is that spherical confinement stabilizes globular proteins because there is no room to unfold.[5] This is ap urely entropic effect because it deals only with the arrangement of molecules.W et est this idea by using the tools of equilibrium thermodynamics to assess the effects of encapsulation in reverse micelles on protein stability.Reverse micelles comprise an aqueous core surrounded by al ayer of surfactant embedded in ab ulk organic solvent (Figure 1). Thespontaneously formed micelles,the inner core diameter of which increases with increasing water loading ratio, [6] have attracted researchers from many fields.Fori nstance,t hey are used as low viscosity protein hosts for protein NMR spectroscopy, [7] to study the properties of confined peptides [8] and proteins, [9] and as model systems for confined water. [10] Protein stability is defined as the modified standard state Gibbs free-energy of unfolding, DG 0 0 u ,w hich equals ÀRTln K u ðÞ ,w here R is the gas constant, T is the absolute temperature,a nd [12b] Dimensions are from the data in Table 1. One reverse micelle contains one protein molecu...

Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin

“…As an example, volume concentration of macromolecules in the cytoplasm of Escherichia coli is as high as 30-40% (3, 31), leading to a significant modulation of macromolecular reaction rates and equilibria. The current consensus is that the functional consequences of molecular crowding stem from two phenomena: hard-core repulsions, otherwise referred to as "entropic effects" (32), and soft chemical interactions (3). In the present paper we demonstrate that the entropic effects are generally more subtle than just the hard-core repulsions, because they necessarily include the phenomenon of forced macromolecule partitioning into protein cavities.…”

Enthalpic stabilization of an SH3 domain by D2O

“…). This shift indicates that SH3 stability is greater in D 2 O at all temperatures and that the effects of D 2 O are mainly enthalpic . , as well as , , and at any temperature can be calculated by fitting the data to Equation .…”

“…71,72 NMR NMR samples were prepared as described. 14,15,71 Briefly, 1 mg of fluorine-labeled, SH3 was resuspended in NMR buffer (50 mM HEPES, bis-tris propane, sodium acetate/acetic acid, pH 7.2) made using H 2 O or 99.9% D 2 O. pH readings are direct measurements and uncorrected for the D 2 O isotope effect. 73 For samples prepared in H 2 O, a coaxial-insert containing D 2 O was used to lock the spectrometer.…”

“…The metastability of this SH3 domain allows stability curves to be constructed and analyzed [13][14][15] at reasonable temperatures (5 C-45 C). The stability curve in D 2 O is simply shifted above the curve in H 2 O (Fig.…”

“…Data were processed as described using Topspin 3.2. 14,15,71 The parameters shown in Table I were calculated using Kirchhoff's equations and the integrated Gibbs-Helmholtz equation as described 14 using MATLAB R2016a.…”