期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：46
页码：16192-16197
DOI：10.1073/pnas.0404549101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We use single-molecule force spectroscopy to drive single GFP molecules from the native state through their complex energy landscape into the completely unfolded state. Unlike many smaller proteins, mechanical GFP unfolding proceeds by means of two subsequent intermediate states. The transition from the native state to the first intermediate state occurs near thermal equilibrium at {approx}35 pN and is characterized by detachment of a seven-residue N-terminal {alpha}-helix from the beta barrel. We measure the equilibrium free energy cost associated with this transition as 22 kBT. Detachment of this small {alpha}-helix completely destabilizes GFP thermodynamically even though the {beta}-barrel is still intact and can bear load. Mechanical stability of the protein on the millisecond timescale, however, is determined by the activation barrier of unfolding the {beta}-barrel out of this thermodynamically unstable intermediate state. High bandwidth, time-resolved measurements of the cantilever relaxation phase upon unfolding of the {beta}-barrel revealed a second metastable mechanical intermediate with one complete {beta}-strand detached from the barrel. Quantitative analysis of force distributions and lifetimes lead to a detailed picture of the complex mechanical unfolding pathway through a rough energy landscape.
