Naganathan Athi N, Muñoz Victor
Department of Chemistry and Biochemistry, University of Maryland , College Park, Maryland 20742, United States.
J Phys Chem B. 2014 Jul 31;118(30):8982-94. doi: 10.1021/jp504261g. Epub 2014 Jul 21.
Downhill folding proteins fold in microseconds by crossing a very low or no free energy barrier (<3 RT), and exhibit a complex unfolding behavior in equilibrium. Such unfolding complexity is due to the weak thermodynamic coupling that exists between the various structural segments of these proteins, and it is manifested in unfolding curves that differ depending on the structural probe employed to monitor the process. Probe-dependent unfolding has important practical implications because it permits one to investigate the folding energy landscape in detail using multiprobe thermodynamic experiments. This type of thermodynamic behavior has been investigated in depth on the protein BBL, an example of extreme (one-state) downhill folding in which there is no free energy barrier at any condition, including the denaturation midpoint. However, an open question is, to what extent is such thermodynamic behavior observed on less extreme downhill folders? Here we perform a multiprobe spectroscopic characterization of the microsecond folder PDD, a structural and functional homologue of BBL that folds within the downhill regime, but is not an example of one-state downhill folding; rather at the denaturation midpoint PDD folds by crossing an incipient free energy barrier. Model-free analysis of the unfolding curves from four different spectroscopic probes together with differential scanning calorimetry reveals a dispersion of ∼9 K in the apparent melting temperature and also marked differences in unfolding broadness (from ∼50 to ∼130 kJ mol(-1) when analyzed with a two-state model), confirming that such properties are also observed on less extreme downhill folders. We subsequently perform a global quantitative analysis of the unfolding data of PDD using the same ME statistical mechanical model that was used before for the BBL domain. The analysis shows that this simple model captures all of the features observed on the unfolding of PDD (i.e., the intensity and temperature dependence of the different spectroscopic signals). From the model we estimate a free energy landscape for PDD in which the maximal thermodynamic barrier (i.e., at the denaturation midpoint) is only ∼0.5 RT, consistent with previous independent estimates. Our results highlight that multiprobe unfolding experiments in equilibrium combined with statistical mechanical modeling provide important insights into the structural events that take place during the unfolding process of downhill proteins, and thus effectively probe the free energy landscape of these proteins.
下坡折叠蛋白通过跨越非常低或不存在的自由能屏障（<3RT）在微秒内完成折叠，并在平衡状态下表现出复杂的去折叠行为。这种去折叠的复杂性源于这些蛋白质不同结构片段之间存在的弱热力学耦合，并且在取决于用于监测该过程的结构探针的去折叠曲线中表现出来。依赖探针的去折叠具有重要的实际意义，因为它允许人们使用多探针热力学实验详细研究折叠能量景观。这种热力学行为已在蛋白质BBL上进行了深入研究，BBL是极端（单态）下坡折叠的一个例子，在任何条件下，包括变性中点，都不存在自由能屏障。然而，一个悬而未决的问题是，在不太极端的下坡折叠蛋白中，这种热力学行为在多大程度上被观察到？在这里，我们对微秒折叠蛋白PDD进行了多探针光谱表征，PDD是BBL的结构和功能同源物，它在 downhill 区域内折叠，但不是单态下坡折叠的例子；相反，在变性中点，PDD通过跨越一个初始自由能屏障进行折叠。对来自四种不同光谱探针的去折叠曲线以及差示扫描量热法进行无模型分析，结果显示表观熔化温度存在约9K的分散，并且在去折叠宽度上也存在显著差异（用双态模型分析时从约50到约130kJ mol(-1)），这证实了在不太极端的下坡折叠蛋白中也观察到了这些性质。我们随后使用之前用于BBL结构域的相同ME统计力学模型对PDD的去折叠数据进行了全局定量分析。分析表明，这个简单的模型捕捉到了在PDD去折叠过程中观察到的所有特征（即不同光谱信号的强度和温度依赖性）。从该模型中，我们估计了PDD的自由能景观，其中最大热力学屏障（即在变性中点）仅约为0.5RT，这与之前的独立估计一致。我们的结果强调，平衡状态下多探针去折叠实验与统计力学建模相结合能为下坡蛋白去折叠过程中发生的结构事件提供重要见解，从而有效地探测这些蛋白质的自由能景观。
