Department of Genetics, Stanford University , 365 Lasuen Street, Littlefield Center, MC2069, Stanford, California 94305, United States.
J Phys Chem B. 2013 Nov 7;117(44):13748-54. doi: 10.1021/jp406925y. Epub 2013 Oct 24.
The nature of the biological free energy scale (ΔGapp), obtained from translocon mediated insertion studies, has been a major puzzle and the subject of major controversies. Part of the problem has been the complexity of the insertion process that discouraged workers from considering the feasible kinetics schemes and left the possible impression that ΔGapp presents some simple partition. Here we extend and clarify our recent analysis of the insertion problem using well-defined kinetics schemes and a free energy profile. We point out that although the rate constants of some steps are far from being obvious, it is essential to consider explicitly such schemes in order to advance in analyzing the meaning of ΔGapp. It is then shown that under some equilibrium conditions the kinetics scheme leads to a simple formula that allows one to relate ΔGapp to the actual free energy of partitioning between the water, the membrane, and the translocon. Other options are also considered (including limits with irreversible transitions that can be described by linear free energy relationships (LFERs)). It is concluded that it is unlikely that a kinetics plus thermodynamic based analysis can lead to a result that identifies ΔGapp with the partition between the membrane and the translocon. Thus, we argue that unless such analysis is presented, it is unjustified to assume that ΔGapp corresponds to the membrane translocon equilibrium or to some other arbitrary definition. Furthermore, we point out that the presumption that it is sufficient to just calculate the PMF for going from the translocon (TR) to the membrane and then to assume irreversible diffusive motion to water and for further entrance to the membrane is not a valid analysis. Overall, we point out that it is important to try to relate ΔGapp to a well-defined kinetics scheme (regardless of the complication of the system) in order to determine whether the energies of inserting positively charged residues to the membrane are related to the corresponding ΔGapp. It is also suggested that deviations from our simple formula for equilibrium conditions can help in identifying and analyzing kinetics barriers.
从易位子介导的插入研究中获得的生物自由能标度（ΔGapp）的本质一直是一个主要的难题，也是主要争议的主题。部分问题在于插入过程的复杂性，这使研究人员不愿考虑可行的动力学方案，并给人留下了ΔGapp 呈现出某种简单分配的印象。在这里，我们使用明确定义的动力学方案和自由能图扩展和澄清我们最近对插入问题的分析。我们指出，尽管一些步骤的速率常数远非显而易见，但明确考虑这些方案对于分析 ΔGapp 的意义至关重要。然后表明，在某些平衡条件下，动力学方案导致一个简单的公式，允许将 ΔGapp 与水、膜和易位子之间实际分配的自由能联系起来。还考虑了其他选择（包括不可逆转变的极限，可以用线性自由能关系（LFERs）来描述）。结论是，基于动力学和热力学的分析不太可能得出将 ΔGapp 与膜和易位子之间分配相关联的结果。因此，我们认为，除非提出这样的分析，否则假设 ΔGapp 对应于膜易位子平衡或其他任意定义是没有道理的。此外，我们指出，假设仅计算从易位子（TR）到膜的 PMF，然后假设不可逆扩散运动到水，并进一步进入膜的假设是不合理的。总体而言，我们指出，尝试将 ΔGapp 与明确定义的动力学方案相关联（无论系统的复杂性如何）以确定将带正电荷的残基插入膜的能量是否与相应的 ΔGapp 相关是很重要的。还建议，偏离我们平衡条件下的简单公式可以帮助识别和分析动力学障碍。
