Liviu Movileanu

Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore.

Here we describe a new type of biosensor element for detecting proteins in solution at nanomolar concentrations. We tethered a 3.4 kDa polyethylene glycol chain at a defined site within the lumen of the transmembrane protein pore formed by staphylococcal α-hemolysin. The free end of the polymer was covalently attached to a biotin molecule. On incorporation of the modified pore into a lipid bilayer, the biotinyl group moves from one side of the membrane to the other, and is detected by reversible capture with a mutant streptavidin. The capture events are observed as changes in ionic current passing through single pores in planar bilayers. Accordingly, the modified pore allows detection of a protein analyte at the single-molecule level, facilitating both quantification and identification through a distinctive current signature. The approach has higher time resolution compared with other kinetic measurements, such as those obtained by surface plasmon resonance.

Kinetics of duplex formation for individual DNA strands within a single protein nanopore

A single oligonucleotide was covalently attached to a genetically engineered subunit of the heptameric protein pore, α-hemolysin, to allow DNA duplex formation inside the pore lumen. Single-channel current recording was used to study the properties of the modified pore. On addition of an oligonucleotide 8 bases in length and with a sequence complementary to the tethered DNA strand, current blockades with durations of hundreds of milliseconds occurred, representing hybridization events of individual oligonucleotides to the tethered DNA strand. Kinetic constants for DNA duplex formation at the single molecule level were derived and found to be consistent with established literature values for macroscopic duplex formation. The resultant equilibrium constant for duplex formation in the nanopore was found to be close to the experimentally derived constant for duplex formation in solution. A good agreement between the equilibrium constants for duplex formation in the nanopore and in solution was also found for two other oligonucleotide pairs. In addition, the nanopore recordings revealed details of the kinetics difficult to obtain by conventional methods, like surface plasmon resonance, which measure ensemble properties. By investigating the temperature dependence of DNA duplex formation at the single molecule level, the standard enthalpy and entropy of the interaction could be obtained.

Interrogating single proteins through nanopores: challenges and opportunities

A single nanopore represents an amazingly versatile single-molecule probe that can be employed to reveal several important features of polypeptides, such as their folding state, backbone flexibility, mechanical stability, binding affinity to other interacting ligands and enzymatic activity. Moreover, groundwork in this area using engineered protein nanopores has demonstrated new opportunities for discovering the biophysical rules that govern the transport of proteins through transmembrane protein pores. In this review, I summarize the current knowledge in the field and discuss how nanopore probe techniques will provide a new generation of research tools in nanomedicine for quantitatively examining the details of complex recognition and, furthermore, will represent a crucial step in designing other pore-based nanostructures and high-throughput devices for molecular biomedical diagnosis.

Controlling a Single Protein in a Nanopore through Electrostatic Traps

Protein-protein pore interaction is a fundamental and ubiquitous process in biology and medical biotechnology. Here, we employed high-resolution time-resolved single-channel electrical recording along with protein engineering to examine a protein-protein pore interaction at single-molecule resolution. The pore was formed by Staphylococcus aureus alpha-hemolysin (alphaHL) protein and contained electrostatic traps formed by rings of seven aspartic acid residues placed at two different positions within the pore lumen. The protein analytes were positively charged presequences (pb2) of varying length fused to the small ribonuclease barnase (Ba). The presence of the electrostatic traps greatly enhanced the interaction of the pb2-Ba protein with the alphaHL protein pore. This study demonstrates the high sensitivity of the nanopore technique to an array of factors that govern the protein-protein pore interaction, including the length of the pb2 presequence, the position of the electrostatic traps within the pore lumen, the ionic strength of the aqueous phase, and the transmembrane potential. Alterations in the functional properties of the pb2-Ba protein and the alphaHL protein pore and systematic changes of the experimental parameters revealed the balance between forces driving the pb2-Ba protein into the pore and forces driving it out.

Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore.

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β‐barrel pore‐forming toxins (β‐PFTs). By contrast, other β‐PFTs form homo‐oligomeric pores; for example, the staphylococcal α‐hemolysin (αHL) pore is a homoheptamer. Here, we deduce the subunit composition of a leukocidin pore by two independent methods: gel shift electrophoresis and site‐specific chemical modification during single‐channel recording. Four LukF and four LukS subunits coassemble to form an octamer. This result in part explains properties of the leukocidin pore, such as its high conductance compared to the αHL pore. It is also pertinent to the mechanism of assembly of β‐PFT pores and suggests new possibilities for engineering these proteins.

Interaction of the antioxidant flavonoid quercetin with planar lipid bilayers

Our capacitance and conductance measurements on reconstituted planar lipid bilayers (BLM) suggest an insertion of the flavonoid quercetin (QCT) in the membranes, which is concentration- and pH-dependent. Interaction of the flavonoid with the membrane has no impact on either structure or integrity of the lipid bilayer. The QCT molecules penetrate the lipid bilayer by intercalating between the flexible acyl chains of the phospholipids, the deepest insertion occuring in acidic medium, when QCT is neutral and completely liposoluble. Results indicated that aggregation of QCT within the hydrophobic core is accompanied by an increase of the transmembrane conductance following an alteration of the hydrophobic barrier for small electrolytes. By contrast, within alkaline media where QCT is deprotonated, the reaction site of the flavonoid is restricted to the hydrophilic domain of the membrane. This significantly changes the double layer capacitance as the negatively charged QCT molecules become sandwiched between polar headgroups at the bilayer surface. At highest alkaline pH, the transmembrane conductance was not affected, since QCT did not perturb the molecular packing of the hydrocarbonic acyl chains of the phospholipids. Results also demonstrated that changes in physical properties of the lipid bilayers following interstitial QCT embedding within either the hydrophobic domain or the polar headgroup domain may be related to both its lipophilic nature and interactions with the electric dipole moments of the polar headgroups of phospholipids. Data also demonstrated that translocation of QCT in the polar part of the lipid bilayer, at physiological pH and salt conditions, may be correlated with its optimized radical scavenging activity. This paper discusses the significance of the free radical scavenging capacity and antioxidant efficiency of QCT.

Single-Molecule Observation of Protein Adsorption onto an Inorganic Surface

Understanding the interactions between silicon-based materials and proteins from the bloodstream is of key importance in a myriad of realms, such as the design of nanofluidic devices and functional biomaterials, biosensors, and biomedical molecular diagnosis. By using nanopores fabricated in 20 nm-thin silicon nitride membranes and highly sensitive electrical recordings, we show single-molecule observation of nonspecific protein adsorption onto an inorganic surface. A transmembrane potential was applied across a single nanopore-containing membrane immersed into an electrolyte-filled chamber. Through the current fluctuations measured across the nanopore, we detected long-lived captures of bovine serum albumin (BSA), a major multifunctional protein present in the circulatory system. Based upon single-molecule electrical signatures observed in this work, we judge that the bindings of BSA to the nitride surface occurred in two distinct orientations. With some adaptation and further experimentation, this approach, applied on a parallel array of synthetic nanopores, holds potential for use in methodical quantitative studies of protein adsorption onto inorganic surfaces.

Partitioning of Individual Flexible Polymers into a Nanoscopic Protein Pore

Polymer dynamics are of fundamental importance in materials science, biotechnology, and medicine. However, very little is known about the kinetics of partitioning of flexible polymer molecules into pores of nanometer dimensions. We employed electrical recording to probe the partitioning of single poly(ethylene glycol) (PEG) molecules, at concentrations near the dilute regime, into the transmembrane beta-barrel of individual protein pores formed from staphylococcal alpha-hemolysin (alphaHL). The interactions of the alpha-hemolysin pore with the PEGs (M(w) 940-6000 Da) fell into two classes: short-duration events (tau approximately 20 micro s), approximately 85% of the total, and long-duration events (tau approximately 100 micro s), approximately 15% of the total. The association rate constants (k(on)) for both classes of events were strongly dependent on polymer mass, and values of k(on) ranged over two orders of magnitude. By contrast, the dissociation rate constants (k(off)) exhibited a weak dependence on mass, suggesting that the polymer chains are largely compacted before they enter the pore, and do not decompact to a significant extent before they exit. The values of k(on) and k(off) were used to determine partition coefficients (Pi) for the PEGs between the bulk aqueous phase and the pore lumen. The low values of Pi are in keeping with a negligible interaction between the PEG chains and the interior surface of the pore, which is independent of ionic strength. For the long events, values of Pi decrease exponentially with polymer mass, according to the scaling law of Daoud and de Gennes. For PEG molecules larger than approximately 5 kDa, Pi reached a limiting value suggesting that these PEG chains cannot fit entirely into the beta-barrel.

Partitioning of a polymer into a nanoscopic protein pore obeys a simple scaling law

The dependence of the rate on polymer mass was examined for the reaction of four sulfhydryl-directed poly(ethylene glycol) reagents with cysteine residues located in the lumen of the staphylococcal α-hemolysin pore. The logarithms of the apparent rate constants for a particular site in the lumen were proportional to N, the number of repeat units in a polymer chain. The proportionality constant was −(a/D)5/3, where a is the persistence length of the polymer (≈3.5Å) and D is the diameter of the pore. Despite some incongruencies with the assumptions of the derivation, the result suggests that the polymers partition into the lumen of the pore according to the simple scaling law of Daoud and de Gennes, cpore/csolution = exp(−N(a/D)5/3). Therefore, the measured reaction rates yield an estimate of the diameter of the pore and might be applied to determine the approximate dimensions of cavities within other similar proteins.

Structural insight into OprD substrate specificity

OprD proteins form a large family of substrate-specific outer-membrane channels in Gram-negative bacteria. We report here the X-ray crystal structure of OprD from Pseudomonas aeruginosa, which reveals a monomeric 18-stranded β-barrel characterized by a very narrow pore constriction, with a positively charged basic ladder on one side and an electronegative pocket on the other side. The location of highly conserved residues in OprD suggests that the structure represents the general architecture of OprD channels.