Vu Dung M, Myers Jeffrey K, Oas Terrence G, Dyer R Brian
Biosciences Division, Mail Stop J586, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Biochemistry. 2004 Mar 30;43(12):3582-9. doi: 10.1021/bi036203s.
Fast relaxation kinetics studies of the B-domain of staphylococcal protein A were performed to characterize the folding and unfolding of this small three-helix bundle protein. The relaxation kinetics were initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The kinetics monitored within the amide I' absorbance of the polypeptide backbone exhibit two distinct kinetics phases with nanosecond and microsecond relaxation times. The fast kinetics relaxation time is close to the diffusion limits placed on protein folding reactions. The fast kinetics phase is dominated by the relaxation of the solvated helix (nu = 1632 cm(-1)), which reports on the fast relaxation of the individual helices. The slow kinetics phase follows the cooperative relaxation of the native helical bundle core that is monitored by both solvated (nu = 1632 cm(-1)) and buried helical IR bands (nu = 1652 cm(-1)). The folding rates of the slow kinetics phase calculated over an extended temperature range indicate that the core formation of this protein follows a pathway that is energetically downhill. The unfolding rates are much more strongly temperature-dependent indicating an activated process with a large energy barrier. These results provide significant insight into the primary process of protein folding and suggest that fast formation of helices can drive the folding of helical proteins.
对葡萄球菌蛋白A的B结构域进行了快速弛豫动力学研究，以表征这种小三螺旋束蛋白的折叠与解折叠过程。弛豫动力学通过激光诱导温度跳跃启动，并使用时间分辨红外光谱进行探测。在多肽主链的酰胺I'吸光度内监测到的动力学表现出两个不同的动力学阶段，弛豫时间分别为纳秒和微秒。快速动力学弛豫时间接近蛋白质折叠反应的扩散极限。快速动力学阶段主要由溶剂化螺旋（ν = 1632 cm⁻¹）的弛豫主导，这反映了各个螺旋的快速弛豫。缓慢动力学阶段则是由天然螺旋束核心的协同弛豫所导致，这可通过溶剂化（ν = 1632 cm⁻¹）和埋藏螺旋红外波段（ν = 1652 cm⁻¹）进行监测。在扩展温度范围内计算得到的缓慢动力学阶段的折叠速率表明，该蛋白的核心形成遵循能量上向下的途径。解折叠速率对温度的依赖性更强，表明这是一个具有大能量屏障的活化过程。这些结果为蛋白质折叠的主要过程提供了重要见解，并表明螺旋的快速形成可以驱动螺旋蛋白的折叠。
