Influence of low pH on the HMGB1 structure.

<p>A) HMGB1 (black circles) and HMGB1ΔC (red circles) at 5 μM concentration were incubated at different pH values (in citrate/citric acid buffer), and the CM variation (ΔCM) was calculated. Because of the small change in ΔCM, even in a very acidic pH, both proteins were also incubated with Gdn.HCl at pH 2.3 and 5.5 M (black triangle for HMGB1 and red triangle for HMGB1ΔC). B) The secondary structure content of 5 μM HMGB1 at neutral pH (black straight lines) and pH 2.3 (black medium-dashed lines) and of HMGB1ΔC at neutral pH (red straight lines) and pH 2.3 (red medium-dashed lines) was monitored by CD at 20 °C. Spectra were converted to molar ellipticity, as described in the Material & Methods section. C) The interaction of bis-ANS and the proteins was assessed by exciting 10 μM probe in a solution containing 5 μM HMGB1 (black circles) or HMGB1ΔC (red circles) at different pH values after a 1-h incubation at 25 °C. For comparison, HMGB1 and HMGB1ΔC were incubated at pH 2.3 in the presence of 5.5 M Gdn.HCl (closed triangles). Normalized spectrum areas were obtained by dividing the spectrum area value of each pH point by the area value at neutral pH.