Nanoscale Protein Pores Modified with PAMAM Dendrimers

Abstract

We describe nanoscale protein pores modified with a single hyperbranched dendrimer molecule inside the channel lumen. Sulfhydryl-reactive polyamido amine (PAMAM) dendrimers of generations 2, 3 and 5 were synthesized, chemically characterized, and reacted with engineered cysteine residues in the transmembrane pore alpha-hemolysin. Successful coupling was monitored using an electrophoretic mobility shift assay. The results indicate that G2 and G3 but not G5 dendrimers permeated through the 2.9 nm cis entrance to couple inside the pore. The defined molecular weight cutoff for the passage of hyperbranched PAMAM polymers is in contrast to the less restricted accessibility of flexible linear poly(ethylene glycol) polymers of comparable hydrodynamic volume. Their higher compactness makes sulfhydryl-reactive PAMAM dendrimers promising research reagents to probe the structure of porous membrane proteins with wide internal diameters. The conductance properties of PAMAM-modified proteins pores were characterized with single-channel current recordings. A G3 dendrimer molecule in the channel lumen reduced the ionic current by 45%, indicating that the hyperbranched and positively charged polymer blocked the passage of ions through the pore. In line with expectations, a smaller and less dense G2 dendrimer led to a less pronounced current reduction of 25%. Comparisons to recordings of PEG-modified pores revealed striking dissimilarities, suggesting that differences in the structural dynamics of flexible linear polymers vs compact dendrimers can be observed at the single-molecule level. Current recordings also revealed that dendrimers functioned as ion-selectivity filters and molecular sieves for the controlled passage of molecules. The alteration of pore properties with charged and hyperbranched dendrimers is a new approach and might be extended to inorganic nanopores with applications in sensing and separation technology.

Effects of PAMAM dendrimers with various surface functional groups and multiple generations on cytotoxicity and neuronal differentiation using human neural progenitor cells

“…EC50 for HaCaT cell line and SW480 cell line was about 30 μg/mL and 20 μM respectively for G4-HN2, compare with hNPCs which was 6.17 μg/mL (Bielawski et al, 2011). Reactions of positively charged nanoparticles with lipid bilayer of cellular membrane may result in nano-scale hole formation in the membrane layers, allowing molecular transport across the membranes (Thiagarajan et al, 2013;Hong et al, 2004Hong et al, , 2006Mecke et al, 2005;Martin et al, 2007;Kelly et al, 2008aKelly et al, , 2008b. Moreover, uptake of positively charged PAMAM-NH 2 of hNPCs was proved in our study (Fig.…”

Dendrimers in Nanoscale Confinement: The Interplay between Conformational Change and Nanopore Entrance

“…The wide distribution of amplitude values in the histogram might reflect the known polydispersity of PAMAM dendrimers. In HPLC analysis of the commercially sourced dendrimer sample, 16 we found a broad and single major peak that confirms the polydispersity but argues against heterogeneities. The average blockade amplitude for each PAMAM generation was acquired using at least three independent recordings with a total of around 10 000 individual events.…”

“…In research, permeating dendrimers of different size are used to probe the lumen of biological and inorganic nanopores, 11 whereas in biomedicine, the interaction influences the transport of encapsulated therapeutics across biological membranes 12,13 or achieves the blocking of pore-forming toxins. 14,15 In materials science, the permanent lodging results in nanoscale filtration devices 16 and reusable catalysts that enclose the dendrimers within nanotubes. 17 Despite these applications, a fundamental understanding of dendrimer behavior under nanoscale confinement is missing.…”

The Surface Location of Individual Residues in a Bacterial S-Layer Protein

“…The small discrepancy between apparent and expected molecular weight is not of concern as the migration of biopolymer conjugates is seldom additive and depends on the position of the cross-link in the biopolymers. 43,44 A dimer, rather than multimeric bands, is expected as the cysteine mutants were mixed with a large excess of unlabeled wt-SbsB, making cross-linking between mutant proteins very unlikely. The interpretation of the 230-kDa band as a photocross-linked dimer was supported by additional studies on the dimer formation of in vivo expressed cysteine mutant A629C.…”