Bottorf Lauren, Rafferty Sophia, Sahu Indra D, McCarrick Robert M, Lorigan Gary A
Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
J Phys Chem B. 2017 Apr 13;121(14):2961-2967. doi: 10.1021/acs.jpcb.7b00626. Epub 2017 Mar 31.
Electron spin echo envelope modulation (ESEEM) spectroscopy was used to distinguish between the local secondary structures of an α-helix and a 3-helix. Previously, we have shown that ESEEM spectroscopy in combination with site-directed spin labeling (SDSL) and H-labeled amino acids (i) can probe the local secondary structure of α-helices, resulting in an obvious deuterium modulation pattern, where i+4 positions generally show larger H ESEEM peak intensities than i+3 positions. Here, we have hypothesized that due to the unique turn periodicities of an α-helix (3.6 residues per turn with a pitch of 5.4 Å) and a 3-helix (3.1 residues per turn with a pitch of 5.8-6.0 Å), the opposite deuterium modulation pattern would be observed for a 3-helix. In this study, H-labeled d-leucine (Leu) was substituted at a specific Leu residue (i) and a nitroxide spin label was positioned 2, 3, and 4 residues away (denoted i+2 to i+4) on an amphipathic model peptide, LRL. When LRL is solubilized in trifluoroethanol (TFE), the peptide adopts an α-helical structure, and alternatively, forms a 3-helical secondary structure when incorporated into liposomes. Larger H ESEEM peaks in the FT frequency domain data were observed for the i+4 samples when compared to the i+3 samples for the α-helix whereas the opposite pattern was revealed for the 3-helix. These unique patterns provide pertinent local secondary structural information to distinguish between the α-helical and 3-helical structural motifs for the first time using this ESEEM spectroscopic approach with short data acquisition times (∼30 min) and small sample concentrations (∼100 μM) as well as providing more site-specific secondary structural information compared to other common biophysical approaches, such as CD.
电子自旋回波包络调制（ESEEM）光谱法用于区分α螺旋和3螺旋的局部二级结构。此前，我们已经表明，ESEEM光谱法结合定点自旋标记（SDSL）和H标记氨基酸（i）可以探测α螺旋的局部二级结构，产生明显的氘调制模式，其中i + 4位置的H ESEEM峰强度通常比i + 3位置大。在这里，我们假设由于α螺旋（每圈3.6个残基，螺距为5.4 Å）和3螺旋（每圈3.1个残基，螺距为5.8 - 6.0 Å）独特的转角周期性，对于3螺旋将观察到相反的氘调制模式。在本研究中，在两亲性模型肽LRL的特定亮氨酸残基（i）处替换H标记的d - 亮氨酸（Leu），并将氮氧化物自旋标记定位在距离该残基2、3和4个残基处（表示为i + 2至i + 4）。当LRL溶解在三氟乙醇（TFE）中时，该肽采用α螺旋结构，或者当掺入脂质体中时形成3螺旋二级结构。与α螺旋的i + 3样品相比，i + 4样品在FT频域数据中观察到更大的H ESEEM峰，而3螺旋则呈现相反的模式。这些独特的模式首次使用这种ESEEM光谱方法，在短数据采集时间（约30分钟）和小样品浓度（约100 μM）下，提供了相关的局部二级结构信息以区分α螺旋和3螺旋结构基序，并且与其他常见的生物物理方法（如圆二色光谱法）相比，提供了更多位点特异性的二级结构信息。
