Data on the role of accessible surface area on osmolytes-induced protein stabilization

This paper describes data related to the research article “Testing the dependence of stabilizing effect of osmolytes on the fractional increase in the accessible surface area on thermal and chemical denaturations of proteins” [1]. Heat- and guanidinium chloride (GdmCl)-induced denaturation of three disulfide free proteins (bovine cytochrome c (b-cyt-c), myoglobin (Mb) and barstar) in the presence of different concentrations of methylamines (sarcosine, glycine-betaine (GB) and trimethylamine-N-oxide (TMAO)) was monitored by [ϴ]222, the mean residue ellipticity at 222 nm at pH 7.0. Methylamines belong to a class of osmolytes known to protect proteins from deleterious effect of urea. This paper includes comprehensive thermodynamic data obtained from the heat- and GdmCl-induced denaturations of barstar, b-cyt-c and Mb.


How data were acquired
Experiments were performed using Jasco spectropolarimeter, Model J-1500-150 (JASCO Corporation, Japan), equipped with Peltier-type temperature controller Data format Raw, Plotted, analyzed Experimental factors All samples and buffers were filtered with 0.22 μm Millipore filters and degassed. Experimental features All CD spectra were recorded at 1 nm band width, temperature scan rate 1°C/min and data was collected at every 0.1°C Data source location Jamia Millia Islamia, New Delhi, India Data accessibility Data are accessible in this article

Value of the data
Methylamines are stabilizing osmolytes. That is, they shift midpoint of denaturation curves to higher C m (midpoint of the GdmCl-induced unfolding transition) and T m (midpoint of the heat-induced unfolding transition). C m and T m increase with increase in concentrations of methylamines.
Stabilization effect of methylamines in terms of ΔG o D (Gibbs free energy change) obtained from GdmCl-induced denaturation studies are found to be more than that from thermal transitions in cases of Mb and barstar.
The stabilizing effect of methylamine against heat-and GdmCl-induced denaturation is same in the case of b-cyt-c.
We have carried out GdmCl-and heat-induced denaturation experiments of barstar, b-cyt-c and Mb in the absence and presence of different concentrations of different methylamine by following the change in [ϴ] 222 (probe for measuring change in secondary structure). Fig. 1 shows GdmCl-induced denaturation curves of Mb, barstar and b-cyt-c in the absence and presence of 0.25 and 0.75 M of each of sarcosine, glycine-betaine and TMAO at pH 7.0 and 25°C. Denaturation of each of protein was found to be reversible in entire range of methylamine concentrations. Each transition curve was measured at least three times, and analyzed for thermodynamic parameters using the Eq. (1). Values of ΔG o D , m g and C m thus obtained are given elsewhere [1].
where y(g) is the observed [θ] 222 at [g], the molar concentration of GdmCl, y N and y D are [θ] 222 values of N and D molecules under the same experimental conditions in which y(g) was measured, ΔG o D is the value of Gibbs free energy change in the absence of the denaturant, m g is the slope (∂ΔG D /∂[g]) T,P , R is the universal gas constant and T is the temperature in Kelvin. It should, however, be noted that the derivation of Eq. (1) assumes that GdmCl-induced denaturation of each protein is a two-state process. Another assumption is that [g]-dependencies of y N (g) and y D (g) are linear (i.e., y N (g) ¼a N þb N [g] and y D (g) ¼ a D þb D [g], where a and b are [g]-independent parameters, and subscripts N and D represent these parameters for the native and denatured protein molecules, respectively.

Heat-induced denaturation studies in the presence and absence of osmolytes
Heat-induced denaturation of Mb, b-cyt-c and barstar in the absence and presence of different concentrations of each osmolyte (sarcosine, TMAO and glycine betaine) were monitored by [θ] 222 at different pH values. Methods for determining the authentic values of thermodynamic parameters from the analysis of thermal denaturation curves of optical properties have already been published [3][4][5]. It should be noted that this analysis assumes that (i) the transition between N and D states of the protein in the absence and presence of each osmolyte is a two-state process, and (ii) structural characteristics of both N and D states are not affected by osmolytes. Each denaturation curve of the protein at a given [methylamine] and pH was analyzed for T m and ΔH m using a non-linear least- where y(T) is the optical property at temperature T (Kelvin), y N (T) and y D (T) are the optical properties of the native and denatured protein molecules at temperature T (Kelvin) and R is the gas constant. As described earlier [3][4][5], in the analysis of the transition curve, it was assumed that a parabolic function describes the dependence of the optical properties of the native and denatured protein Table 1 Thermodynamic parameters associated with the thermal denaturation of myoglobin in the absence and presence of sarcosine, TMAO and GB at different concentrations and pH values.
[Osmolytes] M pH 5.5 pH 5.7 pH 6.0 ΔH m kcal mol À 1 Table 2 Thermodynamic parameters associated with the thermal denaturation of b-cyt-c in the absence and presence of sarcosine, TMAO and GB at different concentrations and pH values.
[Osmolytes] M pH 6.0 pH 6.5 pH 7.5 ΔH m kcal mol À 1 Table 3 Thermodynamic parameters associated with the thermal denaturation of barstar in the absence and presence of sarcosine, TMAO and GB at different concentrations and pH values.
[Osmolytes] M pH 7.5 pH 8.0 pH 9.0  molecules (i.e., y N (T)¼a N þb N Tþ c N T 2 , and y D (T) ¼a D þb D Tþ c D T 2 , where a N , b N , c N , a D , b D , and c D are temperature-independent coefficients). The temperature-independent constant-pressure heat capacity change (ΔC p ) was determined from slope of the linear plot of ΔH m versus T m , using the relation: Using values of T m , ΔH m and ΔC p the value of ΔG D at any temperature T, ΔG D (T), was estimated with the help of Gibbs-Heltmholtz equation: