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：2022
卷号：119
期号：39
DOI：10.1073/pnas.2202779119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Protein function correlates with its structural dynamics. While theoretical approaches to studying protein energy landscapes are well developed, experimental methods that enable probing these landscapes of proteins remain challenging. We used solid-state nanopores to study the translocation behavior of three mutants of a helix bundle protein and quantified the number of energetically accessible conformational states for each mutant. We found that a slower-folding mutant with access to more conformational states translocates faster than a faster-folding mutant with a smaller number of accessible states, suggesting that ease of folding and ease of translocation are at odds in this case.
Translocation of proteins is correlated with structural fluctuations that access conformational states higher in free energy than the folded state. We use electric fields at the solid-state nanopore to control the relative free energy and occupancy of different protein conformational states at the single-molecule level. The change in occupancy of different protein conformations as a function of electric field gives rise to shifts in the measured distributions of ionic current blockades and residence times. We probe the statistics of the ionic current blockades and residence times for three mutants of the
λ
-repressor family in order to determine the number of accessible conformational states of each mutant and evaluate the ruggedness of their free energy landscapes. Translocation becomes faster at higher electric fields when additional flexible conformations are available for threading through the pore. At the same time, folding rates are not correlated with ease of translocation; a slow-folding mutant with a low-lying intermediate state translocates faster than a faster-folding two-state mutant. Such behavior allows us to distinguish among protein mutants by selecting for the degree of current blockade and residence time at the pore. Based on these findings, we present a simple free energy model that explains the complementary relationship between folding equilibrium constants and translocation rates.
关键词：ennanopore biophysicselectric field unfoldingprotein translocationreciprocal relationprotein folding dynamics
