Department of Molecular Biology and Kellogg School of Science and Technology, The Scripps Research Institute, 10550 North Torrey Pines Road TPC6, La Jolla, CA 92037, USA.
J Mol Biol. 2010 Apr 30;398(2):332-50. doi: 10.1016/j.jmb.2010.03.001. Epub 2010 Mar 11.
The thermodynamic hypothesis of Anfinsen postulates that structures and stabilities of globular proteins are determined by their amino acid sequences. Chain topology, however, is known to influence the folding reaction, in that motifs with a preponderance of local interactions typically fold more rapidly than those with a larger fraction of nonlocal interactions. Together, the topology and sequence can modulate the energy landscape and influence the rate at which the protein folds to the native conformation. To explore the relationship of sequence and topology in the folding of beta alpha-repeat proteins, which are dominated by local interactions, we performed a combined experimental and simulation analysis on two members of the flavodoxin-like, alpha/beta/alpha sandwich fold. Spo0F and the N-terminal receiver domain of NtrC (NT-NtrC) have similar topologies but low sequence identity, enabling a test of the effects of sequence on folding. Experimental results demonstrated that both response-regulator proteins fold via parallel channels through highly structured submillisecond intermediates before accessing their cis prolyl peptide bond-containing native conformations. Global analysis of the experimental results preferentially places these intermediates off the productive folding pathway. Sequence-sensitive Gō-model simulations conclude that frustration in the folding in Spo0F, corresponding to the appearance of the off-pathway intermediate, reflects competition for intra-subdomain van der Waals contacts between its N- and C-terminal subdomains. The extent of transient, premature structure appears to correlate with the number of isoleucine, leucine, and valine (ILV) side chains that form a large sequence-local cluster involving the central beta-sheet and helices alpha2, alpha 3, and alpha 4. The failure to detect the off-pathway species in the simulations of NT-NtrC may reflect the reduced number of ILV side chains in its corresponding hydrophobic cluster. The location of the hydrophobic clusters in the structure may also be related to the differing functional properties of these response regulators. Comparison with the results of previous experimental and simulation analyses on the homologous CheY argues that prematurely folded unproductive intermediates are a common property of the beta alpha-repeat motif.
安芬森的热力学假说假定球状蛋白质的结构和稳定性由其氨基酸序列决定。然而，链拓扑结构已知会影响折叠反应，具有优势局部相互作用的基序通常比具有更大非局部相互作用分数的基序折叠得更快。拓扑结构和序列共同作用可以调节能量景观，并影响蛋白质折叠到天然构象的速度。为了探索在折叠βα-重复蛋白中序列和拓扑结构的关系，这些蛋白主要由局部相互作用决定，我们对两个黄素蛋白样的α/β/α三明治折叠成员进行了实验和模拟分析。Spo0F 和 NtrC 的 N 端受体结构域（NT-NtrC）具有相似的拓扑结构，但序列同一性低，能够测试序列对折叠的影响。实验结果表明，这两种响应调节剂蛋白都通过高度结构化的亚毫秒中间体以平行通道折叠，然后才能进入其顺式脯氨酸肽键含有天然构象。对实验结果的全局分析优先将这些中间体置于有活性的折叠途径之外。序列敏感的 Gō 模型模拟得出结论，Spo0F 中折叠的挫折感（对应于非活性途径中间体的出现）反映了其 N 端和 C 端亚结构域之间的范德华相互作用的竞争。瞬态、过早形成的结构的程度似乎与形成涉及中央β-折叠和α2、α3 和α4 螺旋的大序列局部簇的异亮氨酸、亮氨酸和缬氨酸（ILV）侧链的数量相关。在 NT-NtrC 的模拟中未检测到非活性途径物种，这可能反映了其疏水性簇中 ILV 侧链数量减少。疏水簇在结构中的位置也可能与这些响应调节剂的不同功能特性有关。与以前对同源 CheY 的实验和模拟分析结果进行比较表明，过早折叠的无活性中间体是βα-重复基序的共同特性。
