A literature review of the effects of small molecules on the stability of proteins Thermodynamics of protein folding & stability Proteins have the unique ability to spontaneously form a highly ordered, asymmetric and aperiodic three-dimensional conformation, known as the native structure, which is necessary to carry out their unique functions in the cell. This capacity to repeatedly form a specific structure was initially linked to a thermodynamic hypothesis by Anfinsen (Anfinsen 1973). He discovered that the native structure was the protein conformation with the lowest Gibbs free energy under normal physiological conditions i.e. the most thermodynamically favourable. He also found with experiments involving denatured ribonuclease that, under the right conditions, the protein would be “unscrambled” or refolded, which demonstrated the reversibility of protein unfolding, further supporting the thermodynamic hypothesis. Proteins are only marginally stable in their native form due to the very small change in free energy (ΔG) between the denatured and the native state – around -5.15 kcal for most naturally occurring proteins (Pace 1990). The change in enthalpy (ΔH) is highly negative due to hydrogen bond formation during folding, which favours the native state. Although there is a negative, and so favourable, change in entropy (-TΔS) associated with desolvation of the apolar core upon folding, there is also a positive, and so unfavourable, change in entropy (-TΔS) of the protein structure, which results in
Table . Summary of major mechanisms suggested for the action of denaturants on protein stability Denaturant Direct/ indirect interaction Molecular mechanism suggested Experimental or simulation Reference Interactions with hydrophobic surfaces on protein Guanidinium Direct Guanidinium is dehydrated along planar face and lines up flat along hydrophobic surface, weakening hydrophobic effect Simulation (England et al. 2008) Guanidinium Direct Coating of hydrophobic surfaces, weakening hydrophobic effect Simulation (Godawat et al. 2010) Urea Direct & Indirect Urea interacts favourably with hydrophobic side chains and peptide backbone. It also strengthens water-water interactions Simulation (Stumpe & Grubmüller 2007b; Stumpe & Grubmüller 2007a) Urea Direct Transiently aggregates near hydrophobic surfaces Simulation (Zangi et al. 2009) Urea & Guanidinium Direct Direct binding to specific sites on protein normally occupied by water Experimental (Calorimetry) (Makhatadze & Privalov 1992)
Arginine Cytochrome c 3 750 50 mM citrate & 100 mM KCl -5.2 (Taneja & Ahmad 1994)* Arginine IgG Fab domain 3.5 62.5 100 mM glycine 4.6 (Falconer et al. 2011) Arginine Lysozyme 5 250 50 mM citrate, 100 mM KCl -0.9 (Rishi et al. 1998)* Arginine Lysozyme 5 1000 50 mM citrate, 100 mM KCl -6.1 (Rishi et al. 1998)* Arginine Myoglobin 5 250 50 mM citrate, 100 mM KCl -3.8 (Rishi et al. 1998)* Arginine Myoglobin 5 1000 50 mM citrate, 100 mM KCl -10.7 (Rishi et al. 1998)* Arginine RNase A 5 200 50 mM citrate, 100 mM KCl -3.6 (Rishi et al. 1998)* Arginine RNase A 5 1000 50 mM citrate, 100 mM KCl -4.5 (Rishi et al. 1998)* Arginine RNase A 6.7 400 25 mM potassium phosphate, 150 mM KCl -2 (Bowlus & Somero 1979)* Arginine RNase A 7.5 500 40 mM Tris-HCl -1 (Arakawa & Tsumoto 2003)* Arginine RNase A 7.5 1000 40 mM Tris-HCl -3 (Arakawa & Tsumoto 2003)* Arginine RNase A 10 1000 40 mM Tris-HCl -4 (Arakawa & Tsumoto 2003)* Aspartate IgG Fab domain 3.5 62.5 100 mM glycine -1.1 (Falconer et al. 2011) Glutamate IgG Fab domain 3.5 62.5 100 mM glycine -0.9 (Falconer et al. 2011)
Glutamine IgG Fab domain 3.5 62.5 100 mM glycine 3.7 (Falconer et al. 2011) Glycine BSA 6.3 1000 1800 mM GdnHCl 13-27 (Arakawa & Timasheff 1983)* Glycine Cytochrome c 3 1000 50 mM citrate & 100 mM KCl 3.3 (Taneja & Ahmad 1994)* Glycine Lysozyme 6 1000 20 mM NaCl 7 (Arakawa & Timasheff 1985)* Glycine Lysozyme 5.8/6 2000 20 mM citrate & 55 mM NaCl 9 (Santoro et al. 1992) Glycine RNase A 4.2 2000 40 mM acetate 5.5 (Miyawaki et al. 2014)* Glycine RNase A 5.5 2000 40 mM acetate 7.2 (Miyawaki et al. 2014)* Glycine RNase A 6.7 200 25 mM potassium phosphate, 150 mM KCl 0.6 (Bowlus & Somero 1979)* Glycine RNase A 6.7 1000 25 mM potassium phosphate, 150 mM KCl 3.9 (Bowlus & Somero 1979)* Glycine RNase A 5.8/6 2000 20 mM citrate & 55 mM NaCl 8 (Santoro et al. 1992) Histidine Cytochrome c 3 170 50 mM citrate & 100 mM KCl -2 (Taneja & Ahmad 1994)* Histidine IgG Fab domain 3.5 62.5 100 mM glycine 5.8 (Falconer et al. 2011) Histidine Lysozyme 5 150 50 mM citrate, 100 mM KCl -3.8 (Rishi et al. 1998)* Histidine Myoglobin 5 150 50 mM citrate, 100 mM KCl -7.4 (Rishi et al. 1998)*
Histidine RNase A 5 200 50 mM citrate, 100 mM KCl -4.3 (Rishi et al. 1998)* Lysine Cytochrome c 3 700 50 mM citrate, 100 mM KCl 6.5 (Taneja & Ahmad 1994)* Lysine IgG Fab domain 3.5 62.5 100 mM glycine 5.4 (Falconer et al. 2011) Lysine Lysozyme 5 1000 50 mM citrate, 100 mM KCl 6.7 (Rishi et al. 1998)* Lysine Myoglobin 5 500 50 mM citrate, 100 mM KCl 0.2 (Rishi et al. 1998)* Lysine RNase A 5 200 50 mM citrate, 100 mM KCl -2.2 (Rishi et al. 1998)* Lysine RNase A 5 2000 50 mM citrate, 100 mM KCl 4.2 (Rishi et al. 1998)* Lysine RNase A 6.7 200 25 mM potassium phosphate, 150 mM KCl -0.2 (Bowlus & Somero 1979)* Lysine RNase A 6.7 400 25 mM potassium phosphate, 150 mM KCl 0 (Bowlus & Somero 1979)* Octopine RNase A 6.7 200 25 mM potassium phosphate, 150 mM KCl 0 (Bowlus & Somero 1979)* Proline Cytochrome c 3 1000 50 mM citrate & 100 mM KCl 4.3 (Taneja & Ahmad 1994)* Proline IgG Fab domain 3.5 62.5 100 mM glycine 1.4 (Falconer et al. 2011) Proline Lysozyme 6 1000 20 mM NaCl 4 (Arakawa & Timasheff 1985)* Proline RNase A 4.2 2000 40 mM acetate 0.2 (Miyawaki et al. 2014)*
confirm that a number of stabilising cosolutes are preferentially excluded from the surface of BSA, and more specifically that the degree of exclusion corresponds with the cosolute’s ability as an osmoprotectant (Courtenay et al. 2000). The same group also demonstrated preferential interaction of both urea and guanidinium with BSA using the same technique (Courtenay et al. 2001). Table . Preferential interaction data recorded in the literature for a selection of stabilising small molecules and denaturants Cosolute Preferentially bound or excluded Proteins used Experimental technique used Reference Stabilising cosolutes Arginine (1) <0.5 M Neutral, >0.5 M Excluded (2) Excluded (3) Neutral (4) Weakly excluded (1) BSA, Lysozyme, (2) Lysozyme (3) BSA (4) RNase A (1)VPO, Densimetry (2;3;4) High precision densimetry (1)(Schneider & Trout 2009) (2&3)(Kita et al. 1994) (4)(Lin & Timasheff 1996) Betaine Excluded (1) Lysozyme (2;3) BSA (1;2) High precision densimetry (3) VPO (1)(Arakawa & Timasheff 1985) (2)(Arakawa & Timasheff 1983) (3)(Courtenay et al. 2000) Glucose Excluded RNase A, Lysozyme, Chymotrysinogen A, BSA, Ovalbumin High precision densimetry (Arakawa & Timasheff 1982b) Glycerol Excluded (1;2) BSA (2) Lysozyme, (1;2) VPO (2) High (1)(Courtenay et al. 2000)
precision densimetry (2)(Schneider & Trout 2009) Glycine Excluded Lysozyme High precision densimetry (Arakawa & Timasheff 1985) Lactose Excluded RNase A, Lysozyme, Chymotrysinogen A, BSA, Ovalbumin High precision densimetry (Arakawa & Timasheff 1982b) Lysine hydrochloride (1) Excluded (2) Neutral (3) Bound (1)BSA, Lysozyme, (2) Calf brain tubulin (3) RNase A High precision densimetry (1;2)(Arakawa & Timasheff 1984b) (3)(Lin & Timasheff 1996) Lysine glutamate Excluded BSA, Lysozyme High precision densimetry (Kita et al. 1994) Proline Excluded (1) Lysozyme (2) BSA (1) High precision densimetry (2) VPO (1)(Arakawa & Timasheff 1985) (2)(Courtenay et al. 2000) Sarcosine Excluded Lysozyme High precision densimetry (Arakawa & Timasheff 1985) Serine Excluded Lysozyme High precision densimetry (Arakawa & Timasheff 1985) Sucrose Excluded α-chymotrypsin, Chymotrypsinogen, RNase High precision densimetry (Lee & Timasheff 1981) Taurine Excluded (1) Lysozyme (2) BSA High precision densimetry (1)(Arakawa & Timasheff 1985) (2)(Arakawa & Timasheff 1983) TMAO Excluded BSA VPO (Courtenay et al. 2000) Trehalose Excluded (1) BSA (2) RNase A (1) VPO (2) High precision densimetry (1)(Courtenay et al. 2000) (2)(Lin & Timasheff 1996) α-alanine Excluded (1) Lysozyme (2) BSA High precision densimetry (1)(Arakawa & Timasheff 1985) (2)(Arakawa &
Timasheff 1983) Denaturants Urea Bound (1;2) BSA (2) Lysozyme, (1;2) VPO (2) High precision densimetry (1)(Courtenay et al. 2001) (2)(Schneider & Trout 2009) Guanidine hydrochloride (1) Bound (2) Weakly bound (3) Strongly bound (1) BSA (2) RNase A, catalase, (3) Lysozyme, Chymotrypsinogen A, BSA, α-chymotrypsin (1) VPO (2;3) High precision densimetry (1)(Courtenay et al. 2001) (2;3)(Lee & Timasheff 1974) Timasheff and colleagues also discovered a correlation between preferential hydration and a cosolute’s ability to increase the surface tension of water, and inferred that cosolutes’ effect on the surface tension of water was a potential mechanism for protein stabilisation (Lin & Timasheff 1996). This was certainly shown to be true for many cosolutes including sucrose (Lee & Timasheff 1981), lactose, glucose (Arakawa & Timasheff 1982b) and certain stabilising salts (Arakawa & Timasheff 1982a). However, there are exceptions to the rule including an increase in surface tension of water by guanidinium (Breslow & Guo 1990), which means that the mechanism by itself cannot be responsible for the action of all cosolutes on protein stability. When referring to the preferential interaction theory it is worth noting that preferential hydration and protein hydration are separate concepts (Timasheff 2002a). Preferential hydration does not describe changes in the interaction between the protein and the hydration shell, but rather describes that the cosolute in the three component system is absent from the protein hydration shell. This is a concept that is often misconstrued, with many researchers assuming that preferential hydration indicates that the protein has a larger or a dynamically altered hydration shell (Wallace et al. 2015; Smiatek et al. 2012). Although it is often inferred that preferential hydration means that more structured water molecules are present in the hydration
Many of its properties and mechanisms are thought to be responsible for arginine’s action on protein stability and solubility (see section 1.7.1). Lysine Lysine is structurally similar to arginine in that it is a positively charged amino acid with an aliphatic chain in its side chain (Figure 113). It differs to arginine in that it has a longer aliphatic chain capped with an ammonium group rather than a guanidinium group. As explained earlier in the literature review it is commonly used in therapeutic protein formulations as a stabilising excipient, but is not naturally present as an osmolyte in cells (see section 1.3.3.1). This is similar to the situation for arginine, and its lack of use in cells brings to question whether it stabilises proteins. In the literature there are data to suggest it stabilises and data to suggest it destabilises (see Table 14). Figure . Structure of lysine The protein-specific effects of lysine are supported by the fact that it is preferentially excluded from certain proteins, but demonstrates neutral preferential interaction coefficients for others (Arakawa & Timasheff 1984b). Due to the lack of the guanidinium group, lysine forms less cation-π interactions with aromatic residues on proteins than arginine despite having a positively charged side chain at pH 7 (Shah & Shaikh 2015; Crowley & Golovin 2005). Lysine has also shown a lack of self-association in solution, giving support to the hypothesis that arginine stacks in solution via guanidinium-guanidinium interactions (Vondrásek et al. 2009). Histidine & Imidazole Histidine is the third positively charged amino acid, with an imidazole-based side chain (Figure 114). It has three ionisation sites and as such is commonly used as a buffer at pH 5.5-6.5, as well as occasionally being used as a stabilising excipient (Jorgensen et al. 2009). Most experimental data suggests that histidine destabilises proteins (Table 14),
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