Baseline length and automated fitting of denaturation data

Abstract

Abstract:To understand relationships between protein sequence and stability, we often compare data from proteins that differ by the substitution of one amino acid. Frequently, an amino acid change causes the cooperative denaturation transitions to shift to lower temperatures, diminishing the signal from the native state. Here we show that apparent stability changes, Le., the free energy of denaturation, AG,, can also be caused by a deficiency of points in the low temperature end of the transition. In addition, we suggest a method for overcoming this problem. Keywords: baselines; computer fitting; thermodynamic parametersThere are many techniques, including circular dichroism spectropolarimetry (CD), that can probe protein stability as a function of temperature. Investigators often use computer algorithms to fit these signal-versus-temperature plots to a two-state model (Lumry et al., 1966),(1)The application of this model usually involves defining linear baselines for the native and denatured states:where mN and bN are the slope and y-intercept of the native baseline, mD and bD are these values for the denatured baseline, and T is the absolute temperature. By comparing the signal, AT, at any temperature to the native and denatured baselines, the fraction of protein denatured, CY^,^, can be determined:Reprint requests to: Gary J. Pielak, Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290; e-mail: gar-pielak@ unc.edu. The equilibrium constant for denaturation, KD,T, is obtained via Equation 5, and AGD,T is obtained via Equation 6,where R is the gas constant. The slope of a -R In K,.,-versus-T" plot is the van't Hoff enthalpy, AH,,,. The subscript m means that the slope is evaluated at T,,,, the temperature at which KD,T = I . More often, however, Equations 2 through 6 are inserted into Equation 7, and the data are fit directly.In Equation 7 (Elwell & Schellman, 1977), AC,, is the change in heat capacity upon denaturation.Another simple technique for fitting denaturation curves was developed by Breslauer (1995) through manipulation of the van? Hoff equation to yield:For this method, the baselines are determined manually. A line through the steepest part of the transition ( 8 (~/ 8 T )~, is drawn and its slope is used in conjunction with these baselines and T,, to obtain AH,,, .We have observed that baselines from computer fitting often disagree with those selected by hand. To probe this discrepancy, we chose a large CD thermal denaturation data set (76 points, Fig. l), truncated it at various intervals, and determined the effect of data removal on automated baseline selection,

Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes

“…This linear baseline model ( Figure 2) is appropriate for analysis of experimental data obtained by monitoring the CD signal or tryptophan fluorescence emission intensity (26). In this case, baseline derivatives are dβ N /dT = m N and dβ D / dT = m D , and by combining them with eqs 10 and 11, we obtain…”

The sirtuin SIRT6 regulates stress granules formation in C. elegans and in mammals

“…Under the assumption of a two-state transition, nonlinear leastsquares fitting was performed using SigmaPlot 12 (Systat Software) in order to determine apparent midpoints of thermal unfolding. Linear pre-and post-transition baseline corrections were applied as described (Allen and Pielak, 1998). The signal of buffer solution without SIRT6 was ,6 RFU (data not shown).…”

Renal cell carcinoma risk in type 2 von Hippel–Lindau disease correlates with defects in pVHL stability and HIF-1α interactions

“…Intrinsic tryptophane fluorescence was recorded at l ex ¼ 290 nm and l em ¼ 350 nm on a Hitachi F-4500 fluorescence spectrophotometer at 101C. The fluorescence signal was normalized to fraction denatured (F denatured ) according to standard equations (Allen and Pielak, 1998). The free energy of the folded state in the absence of urea ðDG H 2 O Þ, the midpoint of the folding transition (D 50% ), and the m-value were obtained by fitting the transition curve data points to standard equations (Clarke and Fersht, 1993;Killenberg-Jabs et al, 2002 ).…”