Topping Traci B, Gloss Lisa M
School of Molecular Biosciences, Washington State University, Pullman 99164-4660, USA.
J Mol Biol. 2004 Sep 3;342(1):247-60. doi: 10.1016/j.jmb.2004.07.045.
The equilibrium stabilities to guanidinium chloride (GdmCl)-induced denaturation and kinetic folding mechanisms have been characterized for three archael histones: hFoB from the mesophile Methanobacterium formicicum; hMfB from the thermophile Methanothermus fervidus; and hPyA1 from the hyperthermophile Pyrococcus strain GB-3a. These histones are homodimers of 67 to 69 residues per monomer. The equilibrium unfolding transitions, as measured by far-UV circular dichroism (CD) are highly reversible, two-state processes. The mesophilic hFoB is very unstable and requires approximately 1 M trimethyl-amine-N-oxide (TMAO) to completely populate the native state. The thermophilic histones are more stable, with deltaG degrees (H2O) values of 14 and 16 kcal mol(-1) for hMfB and hPyA1, respectively. The kinetic folding of hFoB and hPyA1 are two-state processes, with no detectable transient kinetic intermediates. For hMfB, there is significant development of CD signal in the stopped-flow dead time, indicative of the formation of a monomeric intermediate, which then folds/associates in a single, second-order step to form the native dimer. While the equilibrium stability to chemical denaturation correlates very well with host growth temperature, there is no simple relationship between folding rates and stability for the archael histones. In the absence of denaturant, the log of the unfolding rates correlate with equilibrium stability. The folding/association of the moderately stable hMfB is the most rapid, with a rate constant in the absence of GdmCl of 3 x 10(6) M(-1) s(-1), compared to 9 x 10(5) M(-1) s(-1) for the more stable hPyA1. It appears that the formation of the hMfB burst-phase monomeric ensemble serves to enhance folding efficiency, rather than act as a kinetic trap. The folding mechanism of the archael histones is compared to the folding of other intertwined, segment-swapped, alpha-helical, DNA-binding dimers (ISSADD), including the eukaryotic heterodimeric histones, which fold more rapidly. The importance of monomeric and dimeric kinetic intermediates in accelerating ISSADD folding reactions is discussed.
已对三种古菌组蛋白的氯化胍（GdmCl）诱导变性的平衡稳定性和动力学折叠机制进行了表征：来自嗜温菌甲酸甲烷杆菌的hFoB；来自嗜热菌嗜热栖热菌的hMfB；以及来自超嗜热菌火球菌GB-3a菌株的hPyA1。这些组蛋白是每个单体由67至69个残基组成的同二聚体。通过远紫外圆二色性（CD）测量的平衡去折叠转变是高度可逆的两态过程。嗜温性的hFoB非常不稳定，需要约1 M的三甲基胺-N-氧化物（TMAO）才能完全形成天然态。嗜热组蛋白更稳定，hMfB和hPyA1的ΔG°（H2O）值分别为14和16 kcal mol⁻¹。hFoB和hPyA1的动力学折叠是两态过程，没有可检测到的瞬态动力学中间体。对于hMfB，在停流死时间内CD信号有显著发展，表明形成了单体中间体，然后该中间体以单一的二级步骤折叠/缔合形成天然二聚体。虽然对化学变性的平衡稳定性与宿主生长温度密切相关，但古菌组蛋白的折叠速率和稳定性之间没有简单的关系。在没有变性剂的情况下，去折叠速率的对数与平衡稳定性相关。中等稳定性的hMfB的折叠/缔合最快，在没有GdmCl时的速率常数为3×10⁶ M⁻¹ s⁻¹，而更稳定的hPyA1为9×10⁵ M⁻¹ s⁻¹。似乎hMfB爆发相单体聚集体的形成有助于提高折叠效率，而不是作为动力学陷阱。将古菌组蛋白的折叠机制与其他相互缠绕、片段交换、α-螺旋、DNA结合二聚体（ISSADD）的折叠进行了比较，包括真核异二聚体组蛋白，后者折叠得更快。讨论了单体和二聚体动力学中间体在加速ISSADD折叠反应中的重要性。
