Elucidation of cosolvent effects thermostabilizing water-soluble and membrane proteins

Journal Pre-proof Elucidation of cosolvent effects thermostabilizing water-soluble and membrane proteins

Satoshi Yasuda, Kazuki Kazama, Tomoki Akiyama, Masahiro Kinoshita, Takeshi Murata PII:

S0167-7322(19)35244-4

DOI:

https://doi.org/10.1016/j.molliq.2019.112403

Reference:

MOLLIQ 112403

To appear in:

Journal of Molecular Liquids

Received date:

19 September 2019

Revised date:

9 December 2019

Accepted date:

25 December 2019

Please cite this article as: S. Yasuda, K. Kazama, T. Akiyama, et al., Elucidation of cosolvent effects thermostabilizing water-soluble and membrane proteins, Journal of Molecular Liquids(2018), https://doi.org/10.1016/j.molliq.2019.112403

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© 2018 Published by Elsevier.

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Elucidation of cosolvent effects thermostabilizing water-soluble and membrane proteins

Satoshi Yasudaa−c, Kazuki Kazamaa, Tomoki Akiyamaa, Masahiro Kinoshita,c,* and

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Takeshi Murataa,b,*

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Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan Molecular Chirality Research Center, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan c Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan

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*Corresponding authors. E-mail address: [email protected] (M. Kinoshita). E-mail address: [email protected] (T. Murata).

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ABSTRACT Thermostability of a water-soluble protein becomes higher with an increase in the gain of translational, configurational entropy of the solvent upon protein folding at 298 K. The gain, which is significantly large when the solvent is water, can be made even larger by the cosolvent addition. In an earlier study, we proposed a measure evaluating the enhancement of thermostability achieved by the cosolvent addition. The larger the measure is, the stronger the enhancement is. The measure becomes larger as the total packing fraction of the solvent increases. In this study, we predict that the measure follows the order, sucrose>glucose~mannitol>erythritol>glycerol. On the other hand, the

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addition of 2-propanol, a monohydric alcohol, lowers the thermostability. These theoretical predictions, which are almost independent of the water-soluble protein species, coincide with the experimental observations. We then argue that they are applicable to membrane proteins for which not only the hydrocarbon groups in nonpolar chains of lipid molecules or in surfactant molecules of detergents but also water molecules play essential roles in protein folding. For membrane proteins

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solubilized in detergents, for example, closer packing of water and cosolvent molecules is also accompanied by that of the hydrocarbon groups, enlarging the entropy gain for the hydrocarbon groups upon protein folding and enhancing the thermostability. We experimentally examine the above argument for two representative membrane proteins. It is corroborated for both of them that the thermostability is actually enhanced by the sugar or polyol addition and the degree of

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Keywords: Protein folding Denaturation Solvation Cosolvent effect

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enhancement follows the order described above, and it is lowered by the addition of 2-propanol. The cosolvent effects, especially those leading to enhanced thermostability, can be comprehended for both of water-soluble and membrane proteins within the same theoretical framework.

Entropy Integral equation theory

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1. Introduction Membrane proteins, which play essential roles in cells for sustaining life [1,2], are quite important as drug targets [3]. They are within the lipid bilayer in a biological system and usually solubilized in detergents (i.e., using surfactant molecules) for fundamental studies. Though their long-term storage is necessitated for functional analyses and biochemical applications, it is often hindered by the intrinsic instability of membrane proteins (most of them are significantly less stable than water-soluble proteins). The ligand binding [4], utilization of mutations [5−7], and introduction of fusion proteins [8,9] are generally adopted for thermostabilizing membrane proteins. When the

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thermostability is enhanced, the stability against perturbations other than heating is also made higher. However, the effective ligands, mutations, or fusion proteins are strongly dependent on the membrane protein species. It is desired to develop a stabilization method which is universally applicable to all of the membrane proteins. It is experimentally known that the cosolvent addition can have significantly large effects on the

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structure and properties of a biomolecule in aqueous solution. For example, the thermostability of a water-soluble protein is enhanced or lowered by the cosolvent addition [10−19]. The elucidation of its mechanism enables us to understand the critical roles of solvent environment in protein folding, structural stability, and function. Moreover, from a pragmatic point of view, it is possible to stabilize the structure of the protein and improve its function through the cosolvent effects. Up to now,

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however, membrane proteins have caught much less attention than water-soluble proteins in these directions. This is not surprising because membrane proteins are hardly soluble in aqueous solution. For water-soluble proteins, the cosolvent effects on the thermostability have been investigated mainly by experiments [10−19]. The addition of sugar or polyol as a cosolvent enhances the

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thermostability, and the extent of enhancement follows the order, sucrose>glucose, for sugar and the order, mannitol>erythritol>glycerol, for polyol. For example, the increase in the denaturation temperature Tm by the addition of sucrose at 1 mol/L is larger than that by the addition of glucose at the same concentration. According to the experimental results reported by Back et al. [11], glucose and mannitol share almost the same extent of enhancement. By contrast, the addition of 2-propanol (a monohydric alcohol), for instance, lowers the thermostability [10,14−16]. It is important to note that the cosolvent effects are almost independent of the water-soluble protein species [10−19]. We recently proposed a physical picture of stabilization or destabilization by the cosolvent addition for a water-soluble protein where the translational, configurational entropy of the solvent is treated as the key factor [20,21]. Hereafter, “solvent” signifies water or a water-cosolvent mixture. The presence of a protein generates an excluded volume (EV) which is inaccessible to the centers of solvent molecules. On the other hand, all the solvent molecules in the entire system (not limited to the solvent molecules near the protein) are entropically correlated because the presence of each solvent molecule generates an EV for the other solvent molecules, and we refer to this entropic correlation as the “solvent crowding”. Protein folding is accompanied by a large decrease in the EV due to the protein, leading to a large increase in the total volume available for the translational 3

Journal Pre-proof displacement of solvent molecules in the entire system and to a significant mitigation of the solvent crowding [22−26]. (In our view, to reduce the “water crowding” is a pivotal factor of the hydrophobic effect as argued in the Appendix.) The resulting solvent-entropy gain, in general, becomes larger as the molecular diameter and the total packing fraction of the solvent decreases and increases, respectively [20,21]. When sugar or polyol is added as a cosolvent, the average molecular diameter increases (factor 1) but water and cosolvent molecules become more closely packed due to the cosolvent hydrophilicity (factor 2). Factor 2 dominates for the cosolvents considered in this study [20,21], leading to a larger gain of solvent entropy. We argued that a water-soluble protein becomes

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more thermostable (i.e., Tm becomes higher) with an increase in the solvent-entropy gain calculated at 298 K [20,21]. We then proposed a measure evaluating the enhancement of thermostability achieved by the cosolvent addition. As the cosolvent hydrophilicity increases, the measure becomes larger and the enhancement becomes stronger. We showed that the measure follows the order, sucrose>glucose, for sugar [20] and the order, mannitol>erythritol>glycerol, for polyol [21]. On the

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other hand, it was shown that the addition of 2-propanol, which is rather hydrophobic, lowers the thermostability [21]. In this study, we examine the orders for sugar and polyol all together and suggest the order, sucrose>glucose~mannitol>erythritol>glycerol. We find that this suggestion, which should be independent of the water-soluble protein species, coincide with the experimental observations.

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In a series of works on membrane proteins [27−32], we argued that the entropic effect arising from the translational displacement of hydrocarbon groups (CH2, CH3, and CH) constituting nonpolar chains in the lipid bilayer is essential for stabilizing the protein structure. The lipid bilayer is immersed in water. It is interesting to ask the following question: What if the total packing fraction of water becomes higher (i.e., water molecules are more closely packed)? Our answer is the following.

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The water entropy-effect becomes more essential, the hydrocarbon groups are more closely packed to reduce the EV generated by the lipid bilayer for water molecules, and the membrane protein is more thermostabilized. Hence, the addition of a cosolvent to water can enhance the thermostability of a membrane protein. Of course, a membrane protein solubilized in detergents should be influenced by the cosolvent addition in the same manner, because the protein is within hydrocarbon groups of detergents (see Section 2.7 for more details). In this study, the aforementioned proposition is experimentally corroborated. More specifically, we measure the thermostability of two membrane proteins solubilized using n-dodecyl β-D-maltoside (DDM) as detergents, which are immersed in water or a water-cosolvent mixture. The two membrane proteins are adenosine A2a receptor (A2aR) and thermophilic rhodopsin (TR) [33,34] which are a G-protein coupled receptor (GPCR) and a microbial rhodopsin, respectively. Their properties are substantially different. For example, their apparent midpoint temperatures of denaturation are 55.5 and 87.2°C, respectively (TR is an exceptionally thermostable membrane protein). Nevertheless, the effects of cosolvent addition observed for both of these proteins are qualitatively the same as those for water-soluble proteins. The sugar and polyol addition enhances 4

Journal Pre-proof the thermostability and the degree of enhancement follows the order, sucrose>glucose~mannitol>erythritol>glycerol. The addition of 2-propanol lowers the thermostability. These results are significant in the following respects: (1) Our theoretical treatment considering a membrane protein immersed in a model bulk solvent (see Section 2.7 for more details) is rationalized; (2) we find that water-soluble and membrane proteins share qualitatively the same dependence of cosolvent effects on the cosolvent species; (3) we now have a theory enabling us to elucidate the cosolvent effects for water-soluble and membrane proteins in the same fashion; and (4) the cosolvent addition can be used as a universal way of thermostabilizing a variety of membrane proteins.

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2. Theory

In Sections 2.1 through 2.5, we reorganize our theoretical methods [20,21] for elucidating the

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sugar and alcohol effects on the thermostability of a water-soluble protein. In Section 2.6, we predict that the measure evaluating the enhancement of thermostability follows the order, sucrose>glucose~mannitol>erythritol>glycerol, and this order is independent of the water-soluble protein species. In Sections 2.7 and 2.8, we argue how the theoretical methods and the above order can be applied to a membrane protein.

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2.1. Folding mechanism for a water-soluble protein We first recapitulate the folding mechanism for a water-soluble protein unveiled in our earlier works [22−26,35−38]. Protein folding causes a large loss of protein conformational entropy. The formation of protein intramolecular hydrogen bonds (HBs) is usually regarded as a factor

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overcoming the conformational-entropy loss. In aqueous solution, however, the formation is unavoidably accompanied by the break of protein-water HBs, and the contribution from the latter is considerably larger. The gain of intramolecular van der Waals (vdW) attractive interaction is almost cancelled out by the loss of protein-water vdW attractive interaction. Consequently, protein folding under the physiological condition exhibits a positive change in enthalpy (H0 at 298 K), as manifested by the measurement made by Terazima and coworkers using a novel experimental technique for apoplastocyanin folding [39,40]. The conformational-entropy loss and the positive enthalpy change are surpassed by a large gain of the translational, configurational entropy of water [40]. In other words, protein folding is driven by this water-entropy gain. As illustrated in Fig. 1, the formation of α-helix and β-sheet secondary structures and the close packing of side chains lead to large decreases in the total EV. The secondary structures are advantageous also in the respect that the break of protein-water HBs is compensated by the formation of intramolecular HBs [35−38]. Importantly, the volume of the configurational phase space for water exhibits a large increase and the water crowding is significantly mitigated upon protein folding, leading to a large water-entropy gain (see the Appendix).

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Journal Pre-proof 2.2. Measure of thermostability of a water-soluble protein The enthalpy change upon protein folding H comprises changes in the protein intramolecular, protein-water, and water-water interaction energies which are negative, positive, and negative, respectively. The change in the protein intramolecular interaction energy remains constant against a change in the system temperature T. On the other hand, the sum of the protein-water and water-water interaction energies, which is positive and referred to as the “dehydration energy”, is a decreasing function of T. It is experimentally known that H decreases with increasing T and becomes negative at T=Tm (Tm is the denaturation temperature) [39]. Hence, the thermal denaturation of a protein is

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entropically driven. As argued in our earlier works [41,42] and shown by an experimental study of Fitter [43], the absolute value of the conformational-entropy loss upon protein folding, SC, is an increasing function of T. This is why a protein is denatured at a sufficiently high temperature, i.e., at T=Tm. Let SC,N and SC,D denote the conformational entropies of the native and denatured states, respectively. As

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T increases, the allowed range of a dihedral angle of each amino-acid residue becomes progressively wider, leading to larger SC,D. On the other hand, SC,N exhibits only a small increase due to the constraints caused in the closely packed structure of the protein. As a result, |SC|=|SC,N−SC,D| becomes larger with increasing T. Figure 2 depicts our physical picture for the thermal denaturation, in which Tm is determined by

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the competition of water-entropy and conformational-entropy changes upon protein folding [20,41,42]. We consider three different proteins, proteins 1, 2, and 3 (the solvent is water). A protein comprises significantly many amino-acid residues. There are 20 amino-acid species, and their rates used in the sequence do not largely differ from protein to protein. Therefore, SC,D is approximately proportional to the number of residues Nr. On the other hand, SC,N is far smaller than SC,D. It follows

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that SC/kB (kB is the Boltzmann constant) normalized by Nr, SC/(kBNr), can be considered almost independent of the protein species: SC,3/(kBNr,3)SC,2/(kBNr,2)SC,1/(kBNr,1). As described above, SC/(kBNr) becomes larger with increasing T. We then discuss the water-entropy gain upon protein folding S given by S=SN−SD

(1)

where SN and SD denote the solvation entropies of the native and denatured states, respectively. The solvation entropy is the loss of solvent entropy occurring when a protein in the native or denatured state is inserted into the solvent. Hence, SN and SD are both negative. The protein insertion generates an EV for solvent molecules. Since the EV in the denatured state is larger than that in the native state, |SN||SD|. S is significantly dependent on the details of packing properties of the native state, i.e., the protein species. S increases as the water crowding becomes more significant. It becomes more significant as T becomes higher and water molecules move around more energetically. It apparently becomes less significant as the number density of water  becomes lower. Experimental data on the 6

Journal Pre-proof relation between T and  indicate that above T=298 K,  becomes progressively lower with increasing T. Our theoretical calculation [44] showed that the effect of lowered  is dominant with the result that S (or equivalently, S/(kBNr)) is a weakly decreasing function of T [41,42]. As observed in Fig. 2, as S/(kBNr) at T=298 K increases, Tm becomes higher. Therefore, S/(kBNr) occurring at 298 K can be a reliable measure of the protein thermostability. The incorporation of H/Nr in the physical picture would increase Tm. However, H/Nr is less sensitive to the protein species than SC/(kBNr) [41,42], and the order for the three proteins in Fig. 2, Tm,3Tm,2Tm,1, is not likely to be changed. This is the case as proved in Section 2.6. 2.3. Effects of cosolvent addition on thermostability for a water-soluble protein

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We then consider a protein immersed in three different solvents [20,21]. Solvents α, β, and γ, for instance, correspond to water, water-erythritol mixture, and water-sucrose mixture, respectively (erythritol or sucrose acts as a cosolvent, and the concentrations of erythritol and sucrose share the

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same value). In what follows, “solvent” signifies water or a water-cosolvent mixture. The solvent-entropy gain upon protein folding is significantly influenced by the cosolvent addition. In Fig. 3, the order of the solvent-entropy gain upon folding is ΔSγ>ΔSβ>ΔSα. It is apparent that the order of the denaturation temperature is Tm,γ>Tm,β>Tm,α. The above argument is not justifiable for urea which affects the protein thermostability through

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not only the solvent-entropy effect but also protein-urea vdW attractive interaction. As shown by Matubayasi and coworkers [45] and us [21], the latter factor dominates, with the result that Fig. 3 cannot be applied to the case where urea is considered as a cosolvent. 2.4. Measure of cosolvent effects on thermostability for a water-soluble protein

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On the basis of the discussions in Sections 2.2 and 2.3, we can consider that ∆Σ defined as ∆Σ=∆Smix298/(kBNr)−∆Spw298/(kBNr)

(2)

is a measure of the cosolvent effects [20,21]. Here, ∆Spw is the solvent-entropy gain upon protein folding in water, ∆Smix is that in a water-cosolvent mixture, and the subscript “298” represents that the quantity is evaluated at T=298 K. Positive and negative values of ∆Σ indicate that the cosolvent addition increase and decrease Tm, respectively. A larger value of ∆Σ implies a larger increase or decrease. 2.5. Solvent and protein models for calculating solvation entropy of a water-soluble protein In general, the solvation entropy of a solute S is rather insensitive to the solvent-solvent and solute-solvent interaction potentials. This is because S is determined primarily by the effects of translational freedom of solvent molecules in the entire system, which is more restricted by the solute insertion [23−26,40,45]. Hence, for a solute inserted into water, water can be modeled as a 7

Journal Pre-proof hard-sphere solvent whose particle diameter and packing fraction are set at those of real water, and the solute is modeled as fused hard spheres mimicking its polyatomic structure. The polyatomic structure of the native state of a protein is taken from the experimentally determined three-dimensional structure [20,21]. The denatured state is constructed as an ensemble of sufficiently many (500) random coils [20,21]. A water-cosolvent mixture is modeled as a binary mixture of hard spheres with different sizes [20,21]. The effective particle diameter of a cosolvent (sugar or polyol) is estimated using the experimental density of its solid crystal [46−49]. The volume occupied per cosolvent molecule is

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calculated under the assumption that the crystal possesses the close-packed structure and used in the above estimation [20,21]. For 2-propanol, a different method is adopted. We consider the Stockmayer potential [50] and modify the parameters in its dipole-dipole interaction term, which were evaluated for the gas state, so that the potential can become suitable to the liquid state [21]. Once the particle diameters of water and the cosolvent are determined, the total packing fraction of

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the mixture can be calculated using the experimental density of the water-cosolvent mixture [49,51]. The solvation entropy of a protein with a fixed structure is calculated by a combination of the integral equation theory for the solvation of spherical solutes in simple fluids [52] and our morphometric approach (MA) [53−55]. The MA enables us to take account of the polyatomic structure of the protein with sufficient accuracy and very high speed [54,55]. It was first developed

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for a pure solvent [54] and later extended so that a solvent comprising multicomponents (e.g., a solvent-cosolvent mixture) [55] can also be treated. More details are described in our earlier publications [20,21].

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2.6. Summary of calculation procedure for measure of cosolvent effects on thermostability

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We first consider a hard-sphere solute with diameter dU. The solvent is a water-cosolvent mixture modeled as a binary mixture of hard spheres. The particle diameters of water and the cosolvent are denoted by dS and dC, respectively. The packing fractions of water and the cosolvent are denoted by S and C, respectively. The subscripts “U”, “S”, and “C” denote “solute”, “water”, and “cosolvent”, respectively. The solvation entropy of the solute  comprises two terms determined from the solute-water (US) correlation and the solute-cosolvent (UC) correlation, respectively [55]: =US+UC. In the MA, US or UC is expressed as a linear combination of the four geometric measures defined for the solute-water or solute-cosolvent pair: the EV, water- or cosolvent-accessible surface area, and integrated mean and Gaussian curvatures of the water- or cosolvent-accessible surface [55]. The linear combination is referred to as the “morphometric form”. For the hard-sphere solute, the morphometric form is written as US/kB=C1SC(4πRUS3/3)+C2SC(4πRUS2)+C3SC(4πRUS)+C4SC(4π), RUS=(dU+dS)/2, (3b)

(3a)

UC/kB=C5SC(4πRUC3/3)+C6SC(4πRUC2)+C7SC(4πRUC)+C8SC(4π),

(4a)

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Journal Pre-proof RUC=(dU+dC)/2.

(4b)

The eight coefficients (C1SC−C8SC) are independent of the solute, which is an important idea of the MA. Hence, they are determined beforehand using hard-sphere solutes with different values of dU. Once the eight coefficients are determined, the solvation entropy of a protein with a fixed structure S can readily be calculated as S/kB=SI/kB+SII/kB,

(5a) (5b) (5c)

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SI/kB=C1SCVPS+C2SCAPS+C3SCXPS+C4SCYPS, SII/kB=C5SCVPC+C6SCAPC+C7SCXPC+C8SCYPC.

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Here, V, A, X, and Y are the EV, water- or cosolvent-accessible surface area, and integrated mean and Gaussian curvatures of the water- or cosolvent-accessible surface, respectively. The subscript “PS” or

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“PC” represents that the quantity is calculated for the protein-water or protein-cosolvent pair. The calculation procedure consists of the following steps:

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(A) For sufficiently many different values of dU (the diameter of the hard-sphere solute), the solute-water and solute-cosolvent correlation functions are calculated by numerically solving the

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Ornstein-Zernike and hypernetted-chain (HNC) equations of the integral equation theory for simple fluids [52]. Input parameters for the calculation are dS, dC, S, and C. The robust, efficient algorithm developed by Kinoshita and Harada [56] is used in the numerical solution. (B) US and UC are calculated from the correlation functions using the Morita-Hiroike formula [57,58].

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(C) The eight coefficients (C1SC−C8SC) are determined by the least square fitting to Eqs. (3) and (4). (D) The eight geometric measures (VPS, APS, XPS, YPS, VPC, APC, XPC, and YPC) are calculated using Connolly’s algorithm [59,60]. (E) The solvation entropy of a protein with a fixed structure S is obtained from Eq. (5). When the solvent is water (without a cosolvent), C5SC, C6SC, C7SC, and C8SC are all zero. S calculated for the native structure is SN. SD is obtained as the average of the values of S calculated for the 500 random coils. The following equations hold: ∆Spw=SNpw−SDpw and ∆Smix=SNmix−SDmix where the values calculated for water and the water-cosolvent mixture are denoted by the superscripts “pw” and “mix”, respectively. Though the HNC equation mentioned above is approximate, the errors in SNpw and SDpw or in SNmix and SDmix are cancelled out when the subtraction is made [61]. In Eq. (2), the subscript “298” represents that S and C are estimated at 298 K as described in Section 2.5.

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Journal Pre-proof 2.7. Order of measure of cosolvent effects: Comparison between theoretical predictions and experimental observations for water-soluble proteins We calculate ∆Σ defined by Eq. (2) for protein G for sucrose, glucose, mannitol, erythritol, glycerol, and 2-propanol as the cosolvents by setting the cosolvent concentration at 1 mol/L. The results are compared in Table 1 (the values of dC/dS, S, C, and T=S+C (the total packing fraction of the water-cosolvent mixture) are also given). We note that ∆Σ is roughly in proportion to the cosolvent concentration as observed in experimental studies [10−19]. As observed in Table 1, ∆Σ follows the order, sucrose>glucose~mannitol>erythritol>glycerol. ∆Σ is negative only for 2-propanol.

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When a different protein is considered, the values of ∆Σ change, but their signs and the above order remain unchanged. These theoretical predictions are in good accord with the experimental observations [10−19]. As the cosolvent hydrophilicity becomes higher, water and cosolvent molecules are more closely packed, and the total packing fraction of the water-cosolvent mixture increases, giving rise to more

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significant solvent crowding. Consequently, the water-entropy gain, which originates from the reduction of solvent crowding upon protein folding, becomes larger. This factor plays a dominant role, and ∆Smix298 in Eq. (2) becomes larger for a more hydrophilic cosolvent. We remark that the number of hydroxyl groups in a cosolvent molecule, NHG, follows the orders, “sucrose (8) > glucose (5)” and “mannitol (6) > erythritol (4) > glycerol (3) > 2-propanol (1)” (the number within

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parentheses denotes the value of NHG). As NHG increases, the cosolvent hydrophilicity becomes higher for sugar or polyol. NHG=5 for glucose and 6 for mannitol. However, the molecular structures of glucose and mannitol are substantially different, and it seems that they share almost the same hydrophilicity. Since a 2-propanol molecule possesses a large nonpolar group (CH3CHCH3) and only a single hydroxyl group, 2-propanol is rather hydrophobic and addition to water lowers the total

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packing fraction. We can thus understand the aforementioned order, sucrose>glucose~mannitol>erythritol>glycerol, and the negative value of ∆Σ for 2-propanol. 2.8. Solvent model for a membrane protein In a series of works on membrane proteins [27−32], we pointed out that the hydrocarbon groups (CH2, CH3, and CH) constituting nonpolar chains of the lipid molecules act as the solvent and the entropic factor originating from their translational displacement plays essential roles in the thermostability. In general, the solvent-entropy effects are larger for a smaller particle diameter and a higher packing fraction [20,21]. The particle diameter and the packing fraction of hydrocarbon groups are, on the average, slightly larger and higher than those of water, respectively, and the effects of these differences are compensating [27]. Hence, the hydrocarbon groups can be modeled as hard spheres whose particle diameter and packing fraction are equal to those of water, respectively. A membrane protein comprises transmembrane, extracellular, and intracellular portions. The latter two portions are immersed in water. The transmembrane portion is within the lipid bilayer which is also immersed in water. Taken together, we consider that a membrane protein is present in bulk solvent 10

Journal Pre-proof possessing the particle diameter and the packing fraction pertinent to water. The thermostability of a membrane protein is higher for a larger entropy gain of this solvent upon its folding. Using this concept, we have been successful in a variety of subjects relevant to the thermostabilities of GPCRs and microbial rhodopsins [27−32,62,63]. It was experimentally shown that many membrane proteins fold and oligomerize quite efficiently in nonpolar environments provided by surfactant molecules and amphipols in aqueous solution, which bear little similarity to a membrane [64]. We interpreted this result as follows. First, the specific characteristics of nonpolar chains of lipid molecules are not very important. The entropic

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effect stemming from the translational displacement of hydrocarbon groups common in the surfactant molecules, amphipols, and lipid molecules, play physically the same roles. Second, since the formation of intramolecular HBs in a nonpolar solvent is not accompanied by the break of protein-solvent HBs, the formation itself is a powerful driving force in protein folding. It follows that a membrane protein solubilized in detergents and that within the lipid bilayer share almost the same

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folding mechanism in a physical sense.

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2.9. Effects of cosolvent addition on thermostability for a membrane protein

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When sucrose is added to water as a cosolvent, for example, the packing of water and cosolvent molecules in the water-cosolvent mixture becomes closer than that of water molecules in water. This

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should result in closer packing of the hydrocarbon groups. Namely, the total packing fraction of the solvent for a membrane protein becomes higher, leading to its enhanced thermostability. For a membrane protein, the enthalpy change upon protein folding, H, should be significantly less dependent on T than that for a water-soluble protein. This is because the effect of dehydration energy

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comes into play only for the extracellular and intracellular portions. Taken together, the physical picture illustrated in Fig. 3 should hold true for a membrane protein as well. The effects of cosolvent addition on the thermostability of a membrane protein are most likely to be qualitatively the same as those of a water-soluble protein. For a membrane protein solubilized in detergents, for instance, closer packing of water and cosolvent molecules leads to that of the hydrocarbon groups of detergents as well, enlarging the gain of translational, configurational entropy of the entire solvent upon protein folding, and enhancing the thermostability.

3. Experimental procedure 3.1. Sample preparation A2aR fused with red fluorescent protein (RFP) was expressed in Saccharomyces cerevisiae strain FGY217 cells. The cells were disrupted using glass beads, and membranes were solubilized with 1% n-dodecyl β-D-maltoside (DDM, Anatrace) and 0.2% cholesterol hemisuccinate (CHS, Sigma). DDM works as surfactant molecules. The solubilized A2aR was mixed with TALON metal affinity resin (Clontech) and the bound A2aR was eluted in buffer A (20 mM HEPES, 300 mM imidazole, 11

Journal Pre-proof 10% glycerol, 250 mM NaCl, 0.05% DDM, 0.01% CHS, and 1 g/mL FUT; pH 7.5). The eluted fraction as a purified sample was concentrated to 2 mg/ml using ultrafiltration (ULTRA-4 100 K, Millipore). TR was expressed with 10 μM all-trans-retinal (Sigma) and 0.5 mM isopropyl β-D-1-thiogalactopyranoside (Wako) in Escherichia coli C43 (DE3) cells. The cells were disrupted by sonication, and membranes were solubilized with 1.5% DDM. The solubilized TR was purified by Ni Sepharose 6 Fast Flow (GE Healthcare) and Superdex-200 (GE Healthcare) column chromatographies, after incubations at 70°C for 20 min and at 80°C for 10 min for dissociating the

3.2. Evaluation of thermostability of TR and A2aR

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TR trimers into monomers. The purified TR suspended in buffer B (50 mM Tris-HCl and 0.05% DDM; pH 7.5) was concentrated to 2 mg/ml using ultrafiltration (ULTRA-4 100 K, Millipore) for the thermostability measurement. Our previous publications should be referred to for more detailed information on the expression and purification methods for TR and A2aR [34,65].

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In a previous work [65], we developed a rapid method for measuring the thermostability of a solubilized membrane protein using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with modified Coomassie Brilliant Blue G-250 (mCBB) stain. In this study, we evaluated the

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thermostabilities of TR and A2aR using this method in the presence of 1.0 M cosolvent (sucrose, glucose, mannitol, erythritol, glycerol, or 2-propanol) as explained below. The purified A2aR fused with RFP at the C-terminal shows fluorescence at 595 nm. The thermostability of A2aR can be estimated by monitoring the fluorescence intensities of monomeric

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bands on the CN-PAGE gel. The purified A2aR with a cosolvent was heated at 47°C for 5 min and immediately cooled on ice. The normalized fluorescence intensity was calculated as “fluorescence intensity of the monomeric bands of A2aR fused with RFP after heating” divided by “that before heating” and represented in the percentage. The intensities of the monomeric bands were quantified using ImageJ Software [66]. CN-PAGE was performed using 12.5% Tris-glycine separation gel applied to the treated samples with CN-PAGE buffer (200 mM Tris-HCl, 20% glycerol, 1.0% mCBB, and 1.0% DDM; pH 8.6) at a ratio of 1:1. The A2aR fused with RFP on the gel was visualized (i.e., the gel imaging was performed) using FUSION SOLO 7S (Vilber-Lourmat) after a 5-second exposure to green light at 530 nm with 655 nm cutoff filter. For the determination of the apparent midpoint temperature of denaturation, Tm, a dispensed sample of purified A2aR was heated at a prescribed temperature for 5 min and immediately cooled on ice. We considered sufficiently many different prescribed temperatures in the range 25–80°C. Tm was estimated using GraphPad Prism 4.0 (GraphPad Software) as previously described [65]. The purified TR shows the maximum absorption at 530 nm and is visible in red color whose intensity is strongly correlated with the protein structural properties. Therefore, the thermostability of TR can be estimated by monitoring the decrease in red-color intensity on the CN-PAGE gel. The 12

Journal Pre-proof purified TR with a cosolvent was heated at 91°C for 5 min and immediately cooled on ice. The residual pigment was calculated as “red-color intensity of the monomeric bands of TR after heating” divided by “that before heating” and represented in the percentage. CN-PAGE and the gel imaging were carried out in the same manner as that for A2aR. For determining Tm, a dispensed sample of purified TR was heated at a prescribed temperature for 5 min and immediately cooled on ice. We considered sufficiently many different prescribed temperatures in the range 70–99°C. The estimation of Tm was performed as in the case of A2aR.

4. Results and discussion 4.1. Effects of cosolvent addition on thermostability of A2aR or TR

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The cosolvent effects on the thermostability are illustrated in Fig. 4(a) for A2aR and (b) for TR. The normalized fluorescence intensity represents the percentage of A2aR fused with RFP molecules which were not denatured during the heating at 47°C for 5 min (see Section 3.2). Likewise, the

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residual pigment for TR has physically the same meaning except that the red-color intensity was quantified and the heating was conducted at 91°C. As observed in Fig. 4, the percentage is appreciably influenced by the cosolvent addition. It follows the order, sucrose>glucose>mannitol>erythritol>glycerol>“no cosolvent”>2-propanol, for A2aR and the order, sucrose>glucose~mannitol>erythritol>glycerol>“no cosolvent>2-propanol, for TR. The

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thermostability is largely enhanced by the sucrose, glucose, or mannitol addition. These results are in complete accord with the order experimentally known for water-soluble proteins and theoretically predicted in Table 1. 4.2. Enhanced protein thermostability achieved through mannitol or sucrose addition

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The thermostability of A2aR or TR can also be assessed by measuring the apparent midpoint temperature of denaturation Tm. An example set of images of CN-PAGE gels loaded is shown in Fig. 5(a) for A2aR and (b) for TR. For A2aR, it is observed at 75°C that without a cosolvent the band can hardly be visible, whereas it is considerably more visible when mannitol or sucrose is added. For TR, the band intensity at 95°C disappeared in the absence of a cosolvent, but with mannitol and sucrose added it persisted especially for the sucrose addition. Figure 6 shows an example set of plots of the normalized fluorescence intensity (for A2aR in (a)) or the residual pigment (for TR in (b)) against the temperature. Tm was determined as the inflection point of each curve plotted. The experiments were independently carried out three times, and the average value of Tm was adopted. In Table 2, we collect the values of Tm for A 2aR or TR determined. We find that the increases in Tm for A2aR are 4.4 and 5.5°C with mannitol and sucrose added, respectively, and those for TR are 1.3 and 2.6°C. TR is extremely thermostable even without a cosolvent. In this sense, the amino-acid sequence is almost optimized in TR. Nevertheless, the increase in Tm of TR by the sucrose addition is significant, though it is smaller than that of A2aR.

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Journal Pre-proof 3.3. Significance of the enhanced protein thermostability achieved through cosolvent addition In earlier studies, we found that the binding of ZM241385 (an antagonist for A2aR) [65] and the single mutation S91K [27] increase Tm of A2aR by 5.3 and 7.0°C, respectively. The increase in Tm conferred upon A2aR by the mannitol or sucrose addition at 1 mol/L, 4.4 or 5.5°C, is comparable with the increases due to the antagonist binding and the mutation. Glucose and mannitol should share almost the same increase. Mannitol, glucose, or sucrose acts as an effective stabilizer even for TR, a membrane protein possessing extremely high thermostability without any cosolvent. It should be noted that the increase in Tm becomes larger roughly in proportion to the cosolvent concentration [10−19]. Thus, the cosolvent addition can be a promising means of the thermostability enhancement.

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5. Concluding remarks We previously developed a physical picture of thermal denaturation of a water-soluble protein, which is focused on the two entropic factors, solvent entropy and protein conformational entropy

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[20,21]. The former factor is the configurational entropy originating from the translational displacement of solvent molecules in the entire system. It was proposed that the solvent-entropy gain occurring upon protein folding at 298 K is a measure of the protein thermostability (see Fig. 2). The solvent-entropy gain is significantly large when the solvent is water, but it is made smaller or even larger by adding a cosolvent to water. We argued that ∆Σ defined by Eq. (2) is a measure of the effect

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of cosolvent addition on the protein thermostability (see Fig. 3) [20,21]. ∆Σ can be estimated using a simple solvent model explained in Section 2.5 and a combination of the integral equation theory [52] and the MA extended to a solvent comprising multicomponents [55]. In this study, we calculate the values of ∆Σ for two sugars, three polyols, and 2-propanol (for all

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the cosolvents, the concentration is set at 1 mol/L). We compare them in Table 1. ∆Σ follows the order, sucrose>glucose~mannitol>erythritol>glycerol. ∆Σ is positive for these cosolvents, suggesting that the cosolvent addition enhances the thermostability, and the degree of enhancement follows the above order for the same cosolvent concentration in mol/L. ∆Σ is negative for 2-propanol and this cosolvent lowers the thermostability. Though in Table 1 the estimation is made by taking Protein G as an example, the sign of ∆Σ for each cosolvent and the above order are applicable to any other water-soluble protein. We find that these theoretical predictions are in good agreement with the experimental observations [10−19]. As the cosolvent hydrophilicity, which is correlated primarily with the number of hydroxyl groups in a cosolvent molecule, becomes higher, water and cosolvent molecules are more closely packed than water molecules in water, causing more significant solvent crowding. As a consequence, the water-entropy gain, which originates from the reduction of solvent crowding upon protein folding, becomes larger. This factor plays a dominant role, and ∆Smix298 in Eq. (2) becomes larger for a more hydrophilic cosolvent, leading to higher thermostability. The addition of rather hydrophobic 2-propanol causes the opposite result and lower thermostability. As for a membrane protein either within the lipid bilayer or solubilized in detergents (i.e., surfactant molecules), its relatively more hydrophobic portions are buried in the hydrocarbon groups 14

Journal Pre-proof in nonpolar chains of lipid molecules or surfactant molecules and its rather hydrophilic portions are exposed in water. Thus, a membrane protein is immersed in the hydrocarbon groups and water molecules, either of which act as the solvent. Considering that a membrane protein is immersed in a bulk solvent modeled as explained in Section 2.7, we have been successful in a variety of subjects relevant to the thermostabilities of GPCRs and microbial rhodopsins [27−32,60,61]. When water and cosolvent molecules are more closely packed in a water-cosolvent mixture than water molecules in water, the hydrocarbon groups are also more closely packed, leading to a higher total packing fraction of the bulk solvent. It is probable that the effects of cosolvent addition on the thermostability

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of a membrane protein should be qualitatively the same as those of a water-soluble protein. This proposition has motivated us to experimentally examine the cosolvent effects on the thermostabilities of A2aR and TR which are a GPCR and a microbial rhodopsin, respectively. In what follows, we summarize the experimental findings. For either of the two membrane proteins, the addition of sucrose, glucose, mannitol, erythritol, or glycerol enhances the

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thermostability whereas that of 2-propanol lowers it. The degree of enhancement follows the order, sucrose>glucose~mannitol>erythritol>glycerol, that is exactly the same as that for water-soluble proteins. As discussed in Section 4.3, the increase in Tm (the apparent midpoint temperature of denaturation) conferred upon A2aR by the mannitol, glucose, or sucrose addition at 1 mol/L is significantly large: It is comparable with the increases due to the antagonist binding and the mutation.

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Mannitol, glucose, or sucrose acts as an effective stabilizer even for a membrane protein possessing extremely high thermostability in water such as TR. It should be noted that the increase in Tm becomes larger roughly in proportion to the cosolvent concentration [10−19]. In conclusion, a systematic experimental study on the cosolvent effects for membrane proteins has been performed for the first time. The cosolvent addition can universally enhance the

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thermostability of all of the membrane proteins. Our theory of membrane-protein folding, in which not only the hydrocarbon groups in nonpolar chains of lipid molecules or in surfactant molecules of detergents but also water molecules play crucially important roles as the solvent, has been rationalized. It is now possible to thermostabilize water-soluble and membrane proteins by means of the cosolvent addition in the same fashion. The solvent environments of water-soluble and membrane proteins are apparently different. Furthermore, water-soluble proteins possess a variety of structures with -helices and -sheets in different contents whereas membrane proteins are characterized by the exclusive formation of -helices. Nevertheless, they share the same cosolvent-effect mechanism, which is quite interesting.

Acknowledgments This work was supported by Grant-in-Aid for Scientific Research (No. 17H03663 to M. K., No. 17K15099 to S. Y., and No. 18H05425 to T. M.) from Japan Society for the Promotion of Science (JSPS), by Grant-in-Aid for JSPS fellows, and by Basis for Supporting Innovative Drug Discovery and 15

Journal Pre-proof Life Science Research (BINDS) from Japan Agency for Medical Research and Development (AMED) under Grant Numbers JP18am0101083 and JP19am0101083 (to T. M.).

Appendix: On the hydrophobic effect The hydrophobic effect is one of the most puzzling subjects, and there remain a lot of uncertain and controversial aspects despite a large amount of scientific effort [67–72]. Here, we briefly recapitulate the features of our own theory (refer to our earlier publications [25,26,44,61,73–75] for more details).

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In our opinion, a theory of the hydrophobic effect should be capable of elucidating all of the following facts known for the structures formed by self-assembly processes which are driven by the hydrophobic effect. (1) Experimental studies have shown that the force in forming the structures becomes considerably

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weaker at low temperatures. For example, a protein is unfolded (the cold denaturation) [76], the binding of myosin to filamentous actin (F-actin) is weakened [77], protein aggregate (e.g., amyloid fibril) is dissociated [78,79], and for nonionic amphiphilic molecules the critical micelle concentration becomes higher and the average size of micelles becomes smaller [80]. (2) It is experimentally known that the structures are collapsed by the application of high pressures.

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Typical examples are denaturation of a protein (the pressure denaturation) [81,82], dissociation of F-actin into actin monomers [83], and destruction of amyloid fibril [84]. (3) Experimental studies have shown that the native structure of a protein is stabilized by the addition of sugars and polyols which are hydrophilic, whereas it is destabilized by the addition of rather hydrophobic cosolvents such as 2-propanol [10–19,61]. The addition of salts whose ions are strongly

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hydrated lowers the solubility of a nonpolar solute in water and stabilizes the native structure of a protein [61]. (4) Molecular dynamics simulations have shown that the contact of small molecules, ellipsoids, and plates is entropy driven [85–88]. However, it has been shown with surprise that a model cavity-ligand binding is entropy opposed [89]. Experimentally, receptor-ligand binding processes are often entropy driven, whereas significantly many host-guest complexation processes are entropy opposed [90,91]. In a protein-receptor binding process, as the nonpolar surface area within the binding interface increases, the binding becomes enthalpically more favorable and entropically less favorable [92]. In our theory, the action for reducing the water crowding (entropic correlations among water molecules not only near a solute but also in the bulk) is the hydrophobic effect. We have already been successful in elucidating fact (1) [25,26,74], fact (2) [25,26,73], fact (3) [20,21,61], and fact (4) [75] using our theory where the water crowding is treated as a pivotal factor. An advantage of our theory is that the hydration free energy in the hydrophobic hydration can be decomposed into a 16

Journal Pre-proof variety of physically insightful, energetic and entropic components [20,21,25,26,44,73–75,93,94]. For example, the hydration entropy can be decomposed into the excluded-volume (EV) term and the water-accessible surface term to which the water molecules in the bulk and those near a solute contribute, respectively. Each term can further be decomposed into the solute-water pair correlation component and the solute-water many-body correlation component. The many-body correlation component of the EV term corresponds to the water crowding. Thus, our theory is best suited to the investigation of physical origins of the hydrophobic effect. Apparently, fact (1) indicates that the hydrophobic effect is weakened at low temperatures despite

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the enhanced hydrogen bonds. At low temperatures, the hydrogen bonding of water molecules is strengthened, the translational motion of water molecules becomes less energetic, and water crowding in the entire system is less significant, which leads to the weakened hydrophobicity [94]. Interestingly, fact (2) is ascribed to the severely strengthened hydrophobicity at high pressures [73,95]. The difference between the temperature and pressure effects in terms of the change in degree

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of hydrophobicity is reflected on the difference between the denatured structures of a protein: The cold-denatured structure is significantly extended [96] whereas the pressure-denatured one is just swelling and rather compact [25,26,73]. The crowding effect, where the translational displacement of solvent molecules is a pivotal factor, is present not only in water but also in any fluid solvent. This effect becomes larger as the packing fraction and the molecular diameter become higher and smaller,

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respectively. The present work is relevant to fact (3). We have shown that fact (3) can be elucidated by modeling water as the hard-sphere solvent whose packing fraction and molecular diameter are set at those of water, respectively. A water-cosolvent mixture can also be modeled as a binary mixture of hard spheres with different sizes [20,21,61]. By contrast, it is crucial to employ a realistic molecular model for water in studies on facts (1) and (4). In such cases, the angle-dependent integral equation

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theory (ADIET) has been employed as a quantitatively reliable theory [44,93]. The ADIET is far superior to the reference interaction site model (RISM) and related theories [97,98] for the hydrophobic hydration. (Both of the hard-sphere and molecular models for water give informative results in studies on fact (2).) We briefly summarize the result of our study on fact (4). Entropy opposed (and enthalpy driven) binding occurs when the receptor and the ligand are dominantly nonpolar and a significant amount of space confined between the receptor and ligand surfaces, within which the average number density of water is much lower than that near the isolated receptor or ligand surface, remains even after the binding is completed. This remaining, which is enthalpically necessitated, is unfavorable for the water crowding, causing a loss of water entropy upon binding. Receptors and ligands often comprise polar and charged groups as well as nonpolar groups. In such cases, the ligand-receptor binding interface is closely packed with the achievement of high shape complementarity at the atomic level, with the result that the water-entropy gain arising from the overlap of EVs is dominant. Protein folding is also accompanied by a large gain of water entropy. Refer to Ref. [75] for more details. It is worthwhile to add the following. When the receptor-ligand binding interface is nonpolar, the partial 17

Journal Pre-proof molar volume of the receptor-ligand pair decreases upon binding, with the result that the compression of bulk water occurs under the isobaric condition [99,100]. This compression shifts the water-entropy change in the negative direction, which makes the physical interpretation of the change more difficult. Here, we review the other theories in the literature. There are mainly two types of popular theories. As the factor featuring the hydrophobic effect, one of them suggests an ordered structure of water near a nonpolar group due to the enhanced hydrogen bonding [101–104], and the other (i.e., the Lum-Chandler-Weeks (LCW) theory) emphasizes the dewetting or drying which occurs near a

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nonpolar surface [105]. However, it has been shown that the ordered structure is not formed at the normal temperature (it is formed only at very low temperatures) [44,74,106]. We doubt that the LCW theory is capable of elucidating fact (1) [26,74,75]. According to the LCW theory, the hydration free energy of a nonpolar solute, which is positive, becomes progressively larger with decreasing temperature when the solute size exceeds 1 nm [107]. This result conflicts with the weakening of the

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hydrophobic effect at low temperatures (i.e., fact (1)). On the other hand, for explaining the entropy-opposed binding mentioned in fact (4), one had to suggest that the water structure near a concave, nonpolar surface could be less ordered than the structure of bulk water [89]. However, this suggestion conflicts with the results of statistical-thermodynamics analyses by Hummer and coworkers [108]. In our view, the problem in these theories or interpretations is that they are looking

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at only the water near a solute. As mentioned above, we have shown that facts (1) through (4) can well be elucidated by our theory. To the best of our knowledge, there is no other theory that has been shown to be able to explain all of the four facts. Experimental and computer-simulation studies have shown that a polypeptide made up of 15–25 glycine residues forms a compact structure [109,110]. By analyses on ultra-high resolution structures

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of 178 proteins, it has been found that the average water-accessible surface area is 59% nonpolar, 23% polar, and 19% charged [111]. Thus, it is not necessarily correct to state that nonpolar residues are preferentially buried in protein folding. On the basis of these results, Graziano and coworkers [111] have pointed out that the conventional views of the hydrophobic effect focused on the water structure near a nonpolar group or surface [101–105] should be reconsidered, which is in line with an earlier suggestion made by Harano and Kinoshita [22]. Using all-atom molecular dynamics simulation based on solution theory in energy representation (the ER method), Matubayasi and coworkers have recently performed elaborate studies on protein folding and concluded that they agree with us (the water-crowding effect is referred to as the excluded-volume effect) [112]. The ER method enables us to decompose the hydration free energy into several energetic and entropic components which are physically insightful. As for the studies on fact (3), to study the salting out of a nonpolar solute, Graziano [113,114] calculated the reversible work of cavity creation in water or in alkali-chloride solution using the classical scaled particle theory. He suggested that an increase in the work caused by the addition of an alkali chloride is responsible for the phenomenon. Smith and Mazo [115] studied the salt effects 18

Journal Pre-proof on the solute solubility using the Kirkwood-Buff theory [116] and attributed them to changes in the local solution composition around the solute. Our view is closer to the view of Graziano in the respect that changes in the structure and properties of the bulk solution are more significant.

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Journal Pre-proof Table 1 Values of dC/dS, S, C, and T=S+C used as input data and values of Σ (see Eq. (2) for its definition) estimated for protein Ga. In water without a cosolvent, S=0.3831.

S

C

T

Σ

Sucrose

2.86

0.3021

0.1619

0.4640

0.99

Glucose

2.32

0.3404

0.0864

0.4269

0.43

Mannitol

2.32

0.3371

0.0864

0.4235

0.40

Erythritol

2.06

0.3505

0.0605

0.4110

0.26

Glycerol

1.92

0.3562

0.0490

0.4052

0.19

2-propanol

1.56

0.3561

0.0263

0.3824

−0.08

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dC/dS

The cosolvent concentration is 1 mol/L. We present this result for the first time.

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Cosolvent

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Table 2

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Apparent midpoint temperature of denaturation Tm measured using CN-PAGE for A2aR or TRa. Membrane protein

Tm in case 1

Tm in case 2

Tm in case 3

A2aR

55.5±1.3°C

59.9±1.5°C (4.4°C)

61.0±1.2°C (5.5°C)

87.2±0.4°C

88.5±0.3°C (1.3°C)

89.8±0.6°C (2.6°C)

TR a

No cosolvent is added in case 1, and mannitol and sucrose are added as cosolvents in cases 2 and 3, respectively. The cosolvent concentration is 1 mol/L. The standard error is also given. The number within parentheses indicates the increase in Tm achieved by the cosolvent addition.

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Figure Captions

Fig. 1. (a) Formation of α-helix by a portion of the backbone. (b) Formation of β-sheet by portions of the backbone. (c) Close packing of side chains. Fig. 2. Simple illustration of thermal denaturation of three different proteins (proteins 1, 2, and 3) in water. S and SC denote the solvent-entropy gain and the conformational-entropy loss upon protein

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folding, respectively: In a strict sense, they do not change linearly with increasing T [20,21,42]. Nr, kB, and Tm are the number of residues, Boltzmann constant, and denaturation temperature, respectively. The subscripts, “1”, “2”, and “3”, denote the values for proteins 1, 2, and 3, respectively. SC/(kBNr) represents SC,I/( kBNr,I) (I=1, 2, 3).

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Fig. 3. Simple illustration of thermal denaturation of a protein immersed in three different solvents (solvents α, β, and γ). S and SC denote the solvent-entropy gain and the conformational-entropy loss upon protein folding, respectively: In a strict sense, they do not change linearly with increasing T [20,21,42]. Nr, kB, and Tm are the number of residues, Boltzmann constant, and denaturation temperature, respectively. The subscripts, “α,” “β,” and “γ,” denote the values for the protein in solvents α, β, and γ, respectively.

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Fig. 4. Cosolvent effects on thermal stability of a membrane protein. (a) The membrane protein is A2aR. “Normalized fluorescence intensity” represents the fluorescence intensity of the monomeric bands of A2aR fused with RFP after heating divided by that before heating, which is represented in

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the percentage (see Section 3.2). (b) The membrane protein is TR. “Residual pigment” represents the red-color intensity of monomeric bands of TR after heating divided by that before heating, which is represented in the percentage. In each of (a) and (b), the experiments were independently carried out three times. The standard-error bar is given. Fig. 5. Images of CN-PAGE gels loaded with A2aR (a) and TR (b). The black arrowheads indicate A2aR or TR monomers, and “n.i.” implies that no incubation (heating) is conducted. Fig. 6. Plots of normalized fluorescence intensity for A2aR (a) or residual pigment for TR (b) against temperature. The normalized fluorescence intensity or the residual pigment corresponds to the ratio of the non-denatured A2aR or TR molecules after the heating at a prescribed temperature for 5 min (see Section 3.2). Each data was fitted to a curve of Boltzmann’s sigmoidal equation using GraphPad Prism 4.0, and Tm was determined as the inflection point in the fitted curve. The experiments were independently carried out three times, and the average value of Tm was adopted (see Table 2).

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Credit Author Statement Satoshi Yasuda: Investigation, Validation, Visualization, Writing - Original draft preparation. Kazuki Kazama: Investigation. Tomoki Akiyama: Investigation. Masahiro Kinoshita: Methodology, Formal analysis, Software, Writing – Reviewing and editing. Takeshi Murata: Supervision, Conceptualization, Writing – Reviewing and editing.

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Graphical abstract Highlights

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Cosolvent addition can enhance the thermostability of a protein in aqueous solution. What about a membrane protein solubilized in detergents or within the lipid bilayer?

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Our theory predicts both of the proteins share the same cosolvent-effect mechanism. This prediction is experimentally corroborated for two membrane proteins. It is now possible to thermostabilize different membrane proteins in the same way.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6