Higman Victoria A, Rösner Heike I, Ugolini Raffaella, Greene Lesley H, Redfield Christina, Smith Lorna J
Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, UK.
J Biomol NMR. 2009 Sep;45(1-2):121-31. doi: 10.1007/s10858-009-9342-y. Epub 2009 Jul 19.
Backbone (15)N relaxation parameters and (15)N-(1)H(N) residual dipolar couplings (RDCs) have been measured for a variant of human alpha-lactalbumin (alpha-LA) in 4, 6, 8 and 10 M urea. In the alpha-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala alpha-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. (15)N R(2) values in some regions of all-Ala alpha-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental RDC data are compared with RDCs predicted from a statistical coil model and with bulkiness, average area buried upon folding and hydrophobicity profiles in order to identify regions of non-random structure. Residues in the regions corresponding to the B, D and C-terminal 3(10) helices in native alpha-LA show R(2) values and RDC data consistent with some non-random structural propensities even at high urea concentrations. Indeed, for residues 101-106 the residual structure persists in 10 M urea and the RDC data suggest that this might include the formation of a turn-like structure. The data presented here allow a detailed characterization of the non-cooperative unfolding of all-Ala alpha-LA at higher concentrations of denaturant and complement previous studies which focused on structural features of the molten globule which is populated at lower concentrations of denaturant.
已在4M、6M、8M和10M尿素中测量了人α-乳白蛋白（α-LA）变体的主链（15）N弛豫参数和（15）N-（1）H（N）剩余偶极耦合（RDC）。在α-LA变体中，蛋白质中的八个半胱氨酸残基已被丙氨酸取代（全丙氨酸α-LA）。该蛋白质在pH 2时是部分折叠的熔球态，并且先前已表明在添加尿素时以逐步非协同方式展开。全丙氨酸α-LA某些区域的（15）N R（2）值显示出明显的交换展宽，随着尿素浓度的增加展宽减小。将实验RDC数据与根据统计卷曲模型预测的RDC以及与体积、折叠时埋藏的平均面积和疏水性分布进行比较，以识别非随机结构区域。与天然α-LA中B、D和C末端3（10）螺旋相对应区域的残基即使在高尿素浓度下也显示出与一些非随机结构倾向一致R（2）值和RDC数据。实际上，对于101-106位残基，在10M尿素中仍存在残余结构，RDC数据表明这可能包括形成类似转角的结构。此处提供的数据允许在较高变性剂浓度下对全丙氨酸α-LA的非协同展开进行详细表征，并补充了先前侧重于在较低变性剂浓度下形成的熔球态结构特征的研究。
