Mao Yougang, Ba Yong
Department of Chemistry and Biochemistry, California State University, Los Angeles, 90032, USA.
Biophys J. 2006 Aug 1;91(3):1059-68. doi: 10.1529/biophysj.105.071316. Epub 2006 Apr 28.
The primary sequences of type I antifreeze proteins (AFPs) are Ala rich and contain three 11-residue repeat units beginning with threonine residues. Their secondary structures consist of alpha-helices. Previous activity study of side-chain mutated AFPs suggests that the ice-binding side of type I AFPs comprises the Thr side chains and the conserved i + 4 and i + 8 Ala residues, where i indicates the positions of the Thrs. To find structural evidence for the AFP's ice-binding side, a variable-temperature dependent (13)C spin lattice relaxation solid-state NMR experiment was carried out for two Ala side chain (13)C labeled HPLC6 isoforms of the type I AFPs each frozen in H(2)O and D(2)O, respectively. The first one was labeled on the equivalent 17th and 21st Ala side chains (i + 4, 8), and the second one on the equivalent 8th, 19th, and 30th Ala side chains (i + 6). The two kinds of labels are on the opposite sides of the alpha-helical AFP. A model of Ala methyl group rotation/three-site rotational jump combined with water molecular reorientation was tested to probe the interactions of the methyl groups with the proximate water molecules. Analysis of the T(1) data shows that there could be 10 water molecules closely capping an i + 4 or an i + 8 methyl group within the range of van der Waals interaction, whereas the surrounding water molecules to the i + 6 methyl groups could be looser. This study suggests that the side of the alpha-helical AFP comprising the i + 4 and i + 8 Ala methyl groups could interact with the ice surface in the ice/water interface.
I型抗冻蛋白（AFP）的一级序列富含丙氨酸，并包含三个以苏氨酸残基开头的11个残基的重复单元。它们的二级结构由α螺旋组成。先前对侧链突变AFP的活性研究表明，I型AFP的冰结合侧由苏氨酸侧链以及保守的i + 4和i + 8丙氨酸残基组成，其中i表示苏氨酸的位置。为了找到AFP冰结合侧的结构证据，对分别冷冻在H₂O和D₂O中的I型AFP的两种丙氨酸侧链¹³C标记的HPLC6同工型进行了变温依赖的¹³C自旋晶格弛豫固态NMR实验。第一种在等效的第17和21个丙氨酸侧链（i + 4，8）上标记，第二种在等效的第8、19和30个丙氨酸侧链（i + 6）上标记。这两种标记位于α螺旋AFP的相反两侧。测试了丙氨酸甲基旋转/三位旋转跳跃与水分子重新取向相结合的模型，以探测甲基与邻近水分子的相互作用。对T₁数据的分析表明，在范德华相互作用范围内，可能有10个水分子紧密覆盖i + 4或i + 8甲基，而i + 6甲基周围的水分子可能较松散。这项研究表明，包含i + 4和i + 8丙氨酸甲基的α螺旋AFP侧可能在冰/水界面与冰表面相互作用。
