Quantum Dot-Peptide-Fullerene Bioconjugates for Visualization of in Vitro and in Vivo Cellular Membrane Potential.

We report the development of a quantum dot (QD)-peptide-fullerene (C60) electron transfer (ET)-based nanobioconjugate for the visualization of membrane potential in living cells. The bioconjugate is composed of (1) a central QD electron donor, (2) a membrane-inserting peptidyl linker, and (3) a C60 electron acceptor. The photoexcited QD donor engages in ET with the C60 acceptor, resulting in quenching of QD photoluminescence (PL) that tracks positively with the number of C60 moieties arrayed around the QD. The nature of the QD-capping ligand also modulates the quenching efficiency; a neutral ligand coating facilitates greater QD quenching than a negatively charged carboxylated ligand. Steady-state photophysical characterization confirms an ET-driven process between the donor-acceptor pair. When introduced to cells, the amphiphilic QD-peptide-C60 bioconjugate labels the plasma membrane by insertion of the peptide-C60 portion into the hydrophobic bilayer, while the hydrophilic QD sits on the exofacial side of the membrane. Depolarization of cellular membrane potential augments the ET process, which is manifested as further quenching of QD PL. We demonstrate in HeLa cells, PC12 cells, and primary cortical neurons significant QD PL quenching (ΔF/F0 of 2-20% depending on the QD-C60 separation distance) in response to membrane depolarization with KCl. Further, we show the ability to use the QD-peptide-C60 probe in combination with conventional voltage-sensitive dyes (VSDs) for simultaneous two-channel imaging of membrane potential. In in vivo imaging of cortical electrical stimulation, the optical response of the optimal QD-peptide-C60 configuration exhibits temporal responsivity to electrical stimulation similar to that of VSDs. Notably, however, the QD-peptide-C60 construct displays 20- to 40-fold greater ΔF/F0 than VSDs. The tractable nature of the QD-peptide-C60 system offers the advantages of ease of assembly, large ΔF/F0, enhanced photostability, and high throughput without the need for complicated organic synthesis or genetic engineering, respectively, that is required of traditional VSDs and fluorescent protein constructs.

Secondary Structure Determination of Peptides and Proteins After Immobilization.

The presentation of immobilized peptides and other small biomolecules attached to surfaces can be greatly affected by the attachment chemistry and linking moieties, resulting in altered activity and specificity. For this reason, it is critical to understand how the various aspects of surface immobilization-underlying substrate properties, tether structure, and site of linkage-affect the secondary and quaternary structures of the immobilized species. Here, we present methods for attaching cysteine-containing peptides to quartz surfaces and determining the secondary structure of surface-immobilized peptides. We specifically show that, even when covalently immobilized, changes in peptide conformation can still occur, with measurement occurring in real time.

Application of circular dichroism for structural analysis of surface-immobilized cecropin A interacting with lipoteichoic acid.

The development of biomaterials integrating antimicrobial peptides (AMPs) for improved pathogen detection or use as therapeutic agents requires an understanding of how a peptide may behave once immobilized. Here, we use a combination of circular dichroism and capture assays to assess the structure-function relationship of the cationic amphipathic AMP, cecropin A (cecA), upon interaction with Gram-positive lipoteichoic acids (LTAs). In solution, cecA peptides underwent a change from a largely unstructured conformation in water to structures with significant α-helical content in the presence of both Bacillus subtilis and Staphylococcus aureus LTAs. After surface immobilization, cecA peptides attached by either C- or N-terminus were able to capture both LTAs as well as to undergo conformational changes in the presence of SDS similar to those observed in solution. However, in spite of demonstrated LTA binding activity and the ability to undergo conformational changes (i.e., with SDS), no structural changes were observed when cecA immobilized by its N-terminus was treated with either LTA preparation. On the other hand, cecA immobilized by its C-terminus underwent a conformational change in the presence of S. aureus, but not B. subtilis, LTA. These results indicate that after immobilization recognition of different targets by cationic AMPs may occur by mechanisms quite different from those in solution and that selectivity of these mechanisms is further dependent on the orientation of the immobilized peptide.

Antimicrobial peptide arrays for detection of inactivated biothreat agents.

Arrays of immobilized antimicrobial peptides are used to detect bacterial, viral, and rickettsial pathogens, including inactivated biothreat agents. These arrays differ from the many combinatorial peptide arrays described in the literature in that the peptides used here have naturally evolved to interact with and disrupt microbial membranes with high affinity but broad specificity. The interaction of these naturally occurring peptides with membranes of pathogens has been harnessed for the purpose of detection, with immobilized antimicrobial peptides acting as "capture" molecules in detection assays. Methods are presented for immobilizing the antimicrobial peptides in planar arrays, performing direct and sandwich assays, and detecting bound targets.

Host factors that promote transpososome disassembly and the PriA-PriC pathway for restart primosome assembly.

Initiation of bacteriophage Mu DNA replication by transposition requires the disassembly of the transpososome that catalyses strand exchange and the assembly of a replisome promoted by PriA, PriB, PriC and DnaT proteins, which function in the host to restart stalled replication forks. Once the molecular chaperone ClpX weakens the very tight binding of the transpososome to the Mu ends, host disassembly factors (MRFalpha-DF) promote the dissociation of the transpososome from the DNA template and the assembly of a new nucleoprotein complex. Prereplisome factors (MRFalpha-PR) further alter the complex, allowing PriA binding and loading of major replicative helicase DnaB onto the template promoted by the restart proteins. MRFalpha-PR is essential for DnaB loading by restart proteins even on the deproteinized Mu fork whereas MRFalpha-DF is not required on the deproteinized template. When the transition from transpososome to replisome was reconstituted using MRFalpha-DF and MRFalpha-PR, initiation of Mu DNA replication was strictly dependent upon added PriC and PriA helicase. In contrast, initiation on the deproteinized template was predominantly dependent upon PriB and did not require PriA's helicase activity. The results indicate that transition mechanisms beginning with the transpososome disassembly can determine the pathway of replisome assembly by restart proteins.

Properties of the PriA helicase domain and its role in binding PriA to specific DNA structures.

Primosome assembly protein PriA functions in the assembly of the replisome at forked DNA structures. Whereas its N-terminal DNA binding domain (DBD) binds independently to DNA, the affinity of DBD protein for forked structures is relatively weak. Although the PriA helicase domain (HD) is required for high affinity fork binding, HD protein had very low affinity for DNA. It had only low levels of ATPase activity, and it hydrolyzed ATP when DNA was absent whereas PriA did not. HD catalyzed unwinding of a minimal substrate composed of a duplex with a 3' single-stranded tail. Single-strand binding protein (SSB) bound to the tail of this substrate inhibited this reaction by full-length PriA but enhanced the reaction by HD. SSB stabilized binding of PriA but not of DBD or HD to duplexes with a 5' or 3' single-stranded tail. On forked substrates SSB enhanced helicase action on the lagging-strand arm by PriA but not by HD. The results indicate that synergy of the DBD and HD allows stable binding at the interface between duplex and single-stranded DNA bound by SSB. This mode of binding may be analogous to fork binding, which orients the helicase to act on the lagging-strand side of the fork.