Department of Chemistry, Oakland University, Rochester, MI 48309-4477, USA.
Biopolymers. 2010 Apr;93(4):299-317. doi: 10.1002/bip.21324.
A fluorescently labeled 20-residue polyglutamic acid (polyE) peptide 20 amino acid length polyglutamic acid (E(20)) was used to study structural changes which occur in E(20) as it co-aggregates with other unlabeled polyE peptides. Resonance energy transfer (RET) was performed using an o-aminobenzamide donor at the N-terminus and 3-nitrotyrosine acceptor at the C-terminus of E(20). PolyE aggregates were not defined as amyloid, as they were nonfibrillar and did not bind congo red. Circular dichroism measurements indicate that polyE aggregation involves a transition from alpha-helical monomers to aggregated beta-sheets. Soluble oligomers are also produced along with aggregates in the reaction, as determined through size exclusion chromatography. Time-resolved and steady-state RET measurements reveal four dominant E(20) conformations: (1) a partially collapsed conformation (24 A donor-acceptor distance) in monomers, (2) an extended conformation in soluble oligomers (>29 A donor-acceptor distance), (3) a minor partially collapsed conformation (22 A donor-acceptor distance) in aggregates, and (4) a major highly collapsed conformation (13 A donor-acceptor distance) in aggregates. These findings demonstrate the use of RET as a means of determining angstrom-level structural details of soluble oligomer and aggregated states of proteins.
使用荧光标记的 20 残基聚谷氨酸（polyE）肽（长度为 20 个氨基酸的 polyE（E(20)））来研究 E(20)与其未标记的其他 polyE 肽共同聚集时发生的结构变化。通过 o-氨基苯甲酰胺供体在 E(20)的 N 末端和 3-硝基酪氨酸受体在 C 末端进行共振能量转移（RET）。聚 E 聚集物未被定义为淀粉样蛋白，因为它们是非纤维状的，并且不与刚果红结合。圆二色性测量表明，聚 E 聚集涉及从α-螺旋单体到聚集β-片层的转变。在反应中，除了聚集物外，还会产生可溶性低聚物，这通过排阻色谱法确定。时间分辨和稳态 RET 测量揭示了四种主要的 E(20)构象：（1）单体中部分塌陷的构象（24 A 供体-受体距离），（2）可溶性低聚物中的延伸构象（>29 A 供体-受体距离），（3）聚集物中少量部分塌陷的构象（22 A 供体-受体距离），以及（4）聚集物中主要的高度塌陷构象（13 A 供体-受体距离）。这些发现证明了 RET 可用于确定蛋白质的可溶性低聚物和聚集态的埃级结构细节。
