Quantitative determination of site-specific conformational distributions in an unfolded protein by solid-state nuclear magnetic resonance.
Quantitative determination of site-specific conformational distributions in an unfolded protein by solid-state nuclear magnetic resonance.
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DOI：
10.1016/j.jmb.2009.07.073
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发表时间：
2009-10-02
影响因子：
5.6
通讯作者：
Tycko, Robert
中科院分区：
文献类型：
--
作者：
Hu, Kan-Nian;Havlin, Robert H.;Yau, Wai-Ming;Tycko, Robert
固态核磁共振（NMR）技术被用来调查的35个残基的绒毛蛋白头亚结构域（HP 35）在折叠，部分变性，和完全变性状态的结构。实验在冷冻甘油/水溶液中进行，用盐酸胍（GdnHCl）进行化学变性。在没有GdnHCl的情况下，用均匀的13 C标记所选残基制备的样品的二维固态13 C NMR光谱在化学位移处显示出相对尖锐的交叉，这与折叠的HP 35的已知三螺旋束结构一致。在高浓度的盐酸钆，大多数交叉扩大和移位，定性地表明破坏的折叠结构和静态构象紊乱的发展，在冷冻变性状态。在每个螺旋段中的一个残基的构象分布探测定量与三个固态NMR技术，提供独立的限制与连续对羰基13 C标签的样品中的骨架的α-和β-扭转角。在没有GdnHCl的情况下，组合数据与α-螺旋构象很好地拟合。在[GdnHCl] = 4.5 M时（对应于近似变性中点），合并数据通过每个位点的α-螺旋和部分延伸构象的组合进行良好拟合，但具有位点依赖性群体比。在[盐酸钆] = 7.0 M，对应于完全变性状态，合并的数据很好地拟合的部分扩展和聚脯氨酸II构象的组合，再次与站点依赖的人口比例。两个完全不同的模型的构象分布导致几乎相同的最佳拟合分布，证明了这些结论的鲁棒性。这项工作代表了第一个定量研究的特定位点的构象分布在部分折叠和未折叠状态的蛋白质的固态NMR。
Solid state nuclear magnetic resonance (NMR) techniques are used to investigate the structure of the 35-residue villin headpiece subdomain (HP35) in folded, partially denatured, and fully denatured states. Experiments are carried out in frozen glycerol/water solutions, with chemical denaturation by guanidine hydrochloride (GdnHCl). Without GdnHCl, two-dimensional solid state 13C NMR spectra of samples prepared with uniform 13C labeling of selected residues show relatively sharp crosspeaks at chemical shifts that are consistent with the known three-helix bundle structure of folded HP35. At high GdnHCl concentrations, most crosspeaks broaden and shift, qualitatively indicating disruption of the folded structure and development of static conformational disorder in the frozen denatured state. Conformational distributions at one residue in each helical segment are probed quantitatively with three solid state NMR techniques that provide independent constraints on backbone ϕ and ψ torsion angles in samples with sequential pairs of carbonyl 13C labels. Without GdnHCl, the combined data are well fit by α-helical conformations. At [GdnHCl] = 4.5 M, corresponding to the approximate denaturation midpoint, the combined data are well fit by a combination of α-helical and partially extended conformations at each site, but with a site-dependent population ratio. At [GdnHCl] = 7.0 M, corresponding to the fully denatured state, the combined data are well fit by a combination of partially extended and polyproline II conformations, again with a site-dependent population ratio. Two entirely different models for conformational distributions lead to nearly the same best-fit distributions, demonstrating the robustness of these conclusions. This work represents the first quantitative investigation of site-specific conformational distributions in partially folded and unfolded states of a protein by solid state NMR.
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DOI：
10.1073/pnas.0407146102
发表时间：
2005-02-01
影响因子：
11.1
作者：
Bertoncini, CW;Jung, YS;Zweckstetter, M
通讯作者：
Zweckstetter, M
影响因子：
2.9
作者：
DUKOR, RK;KEIDERLING, TA
通讯作者：
KEIDERLING, TA
影响因子：
3.2
作者：
Dunbar, MG;Novak, BM;Schmidt-Rohr, K
通讯作者：
Schmidt-Rohr, K
影响因子：
4.4
作者：
BENNETT, AE;RIENSTRA, CM;GRIFFIN, RG
通讯作者：
GRIFFIN, RG
DOI：
10.1073/pnas.0502495102
发表时间：
2005-05-24
影响因子：
11.1
作者：
Chiu, TK;Kubelka, J;Davies, DR
通讯作者：
Davies, DR