Advantages of Pyrene Excimer Formation (PEF) over Fluorescence Resonance Energy Transfer (FRET) for Probing the Conformation of Macromolecules in Solution,Macromolecules

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Advantages of Pyrene Excimer Formation (PEF) over Fluorescence Resonance Energy Transfer (FRET) for Probing the Conformation of Macromolecules in Solution
Macromolecules ( IF 5.1 ) Pub Date : 2024-08-28 , DOI: 10.1021/acs.macromol.4c01069
Sanjay Patel ₁ , Jean Duhamel ₁

Affiliation

Pyrene excimer formation (PEF) was applied to study the homopolypeptides poly(d,l-glutamic acid) (PDLGA) and poly(l-glutamic acid) (PLGA), collectively referred to as the PGA samples, in N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). The polypeptides were labeled with 1-pyrenebutylamine (PyC4N) or 1-pyreneoctylamine (PyC8N) and were described as PyCX(x)N-PGA where X equals 4 or 8, respectively, and x represents the mole percentage of pyrene-labeled glutamic acid (Glu). The fluorescence results obtained with these polypeptides were compared with the results obtained earlier with the same polypeptides randomly labeled with 1-pyrenemethylamine (PyC1N). Analysis of the fluorescence decays of the PyCX(x)N-PGA samples with the fluorescence blob model (FBM) yielded the average number (⟨N_blob^exp⟩) of Glu units per blob, where a blob is the volume probed by an excited pyrenyl label. The ⟨N_blob^exp⟩ values obtained for the PyCX(x)N-PGA samples in DMF and DMSO were compared with the N_blob^MMO values determined via molecular mechanics optimizations (MMO) conducted for PLGA constructs adopting four different conformations and labeled with either PyC1N, PyC4N, or PyC8N. The good agreement obtained between the ⟨N_blob^exp⟩ and N_blob^MMO values for PDLGA in DMF and DMSO and PLGA in DMF, whose conformation was known to be a random coil and an α–helix, respectively, validated the PEF-based methodology. It was then applied to determine the unknown conformation of PLGA in DMSO. The excellent match found between ⟨N_blob^exp⟩ obtained for PLGA in DMSO and N_blob^MMO obtained for a PLGA 3₁₀ helix led to its unambiguous assignment as the conformation of PLGA in DMSO. Since PyC8N probed a volume whose 7.7 nm diameter was almost double that of PyC1N, these experiments significantly extended the range of distances over which PEF can be applied to characterize macromolecular conformations, making PEF competitive with the distance range accessible by fluorescence resonance energy transfer (FRET).

中文翻译：

芘准分子形成 (PEF) 相对于荧光共振能量转移 (FRET) 探测溶液中大分子构象的优势

应用芘准分子形成 (PEF) 研究N , N-二甲基甲酰胺中的均聚肽聚( d , l-谷氨酸) (PDLGA) 和聚( l-谷氨酸) (PLGA)（统称为 PGA 样品） (DMF) 和二甲基亚砜 (DMSO)。这些多肽用1-芘丁胺(PyC4N)或1-芘辛胺(PyC8N)标记，并被描述为PyCX( x )N-PGA，其中X分别等于4或8， x代表芘标记的谷氨酸的摩尔百分比（谷氨酸）。将这些多肽获得的荧光结果与之前用1-芘甲胺(PyC1N)随机标记的相同多肽获得的结果进行比较。使用荧光斑点模型 (FBM) 对 PyCX( x )N-PGA 样品的荧光衰变进行分析，得出每个斑点的 Glu 单位的平均数 (⟨ N _blob ^exp ⟩)，其中斑点是由激发态探针探测的体积。芘基标签。将在 DMF 和 DMSO 中的 PyCX( x )N-PGA 样品获得的 ⟨ N _blob ^exp ⟩ 值与通过分子力学优化 (MMO) 确定的N _blob ^MMO值进行比较，该 PLGA 结构采用四种不同的构象并用任一标记PyC1N、PyC4N 或 PyC8N。 DMF 中的 PDLGA 和 DMF 中的 DMSO 和 PLGA 的 ⟨ N _blob ^exp ⟩ 和N _blob ^MMO值之间获得了良好的一致性，其构象分别为无规卷曲和 α-螺旋，验证了基于 PEF 的方法。然后用于确定 DMSO 中 PLGA 的未知构象。在 DMSO 中为 PLGA 获得的 ⟨ N _blob ^exp ⟩ 与为 PLGA 3 ₁₀螺旋获得的N _blob ^MMO之间发现的出色匹配导致其明确指定为 DMSO 中 PLGA 的构象。由于 PyC8N 探测的体积为 7.7 nm 直径，几乎是 PyC1N 的两倍，因此这些实验显着扩展了 PEF 可用于表征大分子构象的距离范围，使 PEF 与荧光共振能量转移 (FRET) 可达到的距离范围相竞争。）。

更新日期：2024-08-28

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