2013年5月21日Biophysical Journal期刊封面亮点

相关专题

2013年5月21日Biophysical Journal期刊封面图片艺术描绘了Pfk在-2-3°C条件下冷变性过程中的发生的分离和溶剂渗透。通过骨干酰胺氢氘交换和质谱法对这个过程进行解释，图片中两侧代表两个质谱图随着时间不断变化。在图片底部水状滴剂包围的肿大亚基表示冷变性过程的终结状态：一个溶剂渗透单体。彩色设计补充表示酰胺类交换范围，通过这个实验为这个区域进行定量。

原文摘要：

Observation of Solvent Penetration during Cold Denaturation of E. coli Phosphofructokinase-2

César A. Ramírez-Sarmiento, Mauricio Baez, Christian A.M. Wilson, Jorge Babul, Elizabeth A. Komives ａnd Victoria Guixé

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N22I mechanism with dimer dissociation ａnd formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region ａnd equaLED the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation ａnd formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner.