Fig 3.

The pH dependence of protein stability of the drkN SH3 domain between pH 1 and pH 7. Experimentally determined values of ΔG_FU (black circles) were determined as ΔG_FU = −RT ln(p_U/p_F), where p_U and p_F are fractional populations as measured from peak volumes of the U_exch and F_exch states in heteronuclear single quantum coherence experiments and error bars represent SD. The calculated pH dependence of ΔG_FU (black line) was derived by using experimental, residue-specific pK_a values for both states employing Eqs. 2 and 3. The vertical offset of the calculated curve, ΔG, was determined by minimizing the χ² with the experimental data. A Monte Carlo simulation was performed by normally distributing pK_a values within their SD as estimated from experimental uncertainties (yellow lines). For comparison, pH stability profiles representing simplified models for the unfolded state are plotted assuming (i) standard pK_a values (blue line) and (ii) general downward shifted pK_a values (by 0.3 pH units from standard values, red line) for the unfolded state but by using experimental pK_a values for the folded state.