Ultrafast Protein Folding

超快蛋白质折叠

• 批准号: 8349693
• 负责人: William Eaton
• 金额: $ 35.1万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349693
• 关键词: Agreement; Amino Acids; Computer Simulation; Data; Detection; Equilibrium; Fluorescence; Guanidinium Chloride; Kinetics; Lasers; Measures; Methods; Molecular Conformation; Monitor; Norleucine; Population; Property; Proteins; Techniques; Theoretical model; Time; Tryptophan; molecular dynamics; mutant; protein folding; research study; simulation; temperature jump; triplet state; villin

Determining the rate of forming the truly folded conformation of ultrafast folding proteins is an important issue for both experiments and simulations. The double-norleucine mutant of the 35-residue villin subdomain is the focus of recent computer simulations with atomistic molecular dynamics because it is currently the fastest folding protein. The folding kinetics of this protein have been measured in laser temperature-jump experiments using tryptophan fluorescence as a probe of overall folding. The conclusion from the simulations, however, is that the rate determined by fluorescence is significantly larger than the rate of overall folding. We have therefore employed an independent experimental method to determine the folding rate. The decay of the tryptophan triplet-state in photoselection experiments was used to monitor the change in the unfolded population for a sequence of the villin subdomain with one amino acid difference from that of the laser temperature-jump experiments, but with almost identical equilibrium properties. Folding times obtained in a two-state analysis of the results from the two methods at denaturant concentrations varying from 1.56.0 M guanidinium chloride are in excellent agreement, with an average difference of only 20%. Polynomial extrapolation of all the data to zero denaturant yields a folding time of 220 (+100,-70) ns at 283 K, suggesting that under these conditions the barrier between folded and unfolded states has effectively disappeared the so-called downhill scenario.

确定形成超快折叠蛋白的真正折叠构象的速率是实验和模拟的重要问题。 35个残留的villin子域的双甲状腺素突变体是最近具有原子分子动力学的计算机模拟的重点，因为它目前是最快的折叠蛋白。该蛋白质的折叠动力学已在激光温度跳跃实验中测量，使用色氨酸荧光作为整体折叠的探针。然而，来自模拟的结论是，荧光确定的速率明显大于整体折叠率。因此，我们采用了一种独立的实验方法来确定折叠率。在照片选择实验中，色氨酸三重态的衰减用于监测一系列Villin子域的序列变化，其氨基酸与激光温度跳跃实验的变化，但具有几乎相同的平衡性能。在两种状态分析中，从1.56.0 m氯化物的两种方法中获得的两种方法的折叠时间非常吻合，平均差异仅为20％。将所有数据的多项式外推到零变性剂在283 K时的折叠时间为220（+100，-70）ns，这表明在这些条件下，折叠状态和展开状态之间的障碍实际上消失了所谓的下坡场景。