文摘
The rigidity and defined length of the polyproline type II helix (PPII) have made it the structural basis of a nanoscopic ruler, which has been widely applied in F枚rster resonance energy transfer (FRET) studies. A growing body of data, however, has questioned the foundation for this and has provided evidence for structural perturbations to the PPII caused by temperature, salt content, solvent polarity, and even Pro repeat length. Here, we examine the polyproline ruler in the context of semiconductor quantum dots (QDs) and FRET. For this study, a series of polyproline peptides (Pron, n = 0, 3, 6, 9, 12, 15, 18) displaying a C-terminal hexahistidine sequence (His6) and an N-terminal cysteine for site-specific labeling with Cy3 dye were synthesized. Peptide rigidity was first examined with ATTO 647 Ni2+-nitrilotriacetic acid acceptor dye coordinated to the His6-termini of the Cy3 donor-labeled peptides. These conditions provided a steady-state fluorescent response that closely followed FRET predictions derived from the expected donor鈥揳cceptor distances; this confirmed the PPII conformation and nanoscopic ruler in the context of our sequences. Peptides were next assembled to negatively charged dihydrolipoic acid functionalized 530 nm emitting QDs, which then acted as a donor to the Cy3 acceptor. These data revealed decreases in FRET efficiency E but at significantly less than the magnitude predicted. Lastly, peptides were assembled to neutral poly(ethylene glycol) or PEG-appended dihydrolipoic acid functionalized 530 nm QDs, and here FRET E did not change as peptide length increased. The latter observations were confirmed with excited-state lifetime measurements and single-pair FRET analysis. Circular dichroism spectroscopy was performed on select peptides both free in solution and as assembled to the PEGylated QDs along with physical characterization by dynamic light scattering and electrophoretic mobility. Overall, analysis confirms the initial validity of the rigid polyproline ruler, while it also suggests that peptide subpopulations adopt a different conformation when attached to QDs. Rather than a gross structural rearrangement, this change is consistent with a trans to cis bond reversion in some of the Pro鈥揚ro peptidyl bond(s), which alters persistence length. This suggests that the PPII is highly context dependent and can be strongly influenced by microenvironments or interfacial effects and thus requires careful consideration of experimental format and related factors before being implemented with nanoparticles.
