Unzipping of Double-stranded DNA in Engineered α-Hemolysin Pores

Biological protein α-hemolysin nanopore is under intense investigation as a potential platform for rapid and low-cost DNA sequencing. However, due to its narrow constriction, analysis of DNA in the α-hemolysin pore has long time been restricted to single strands. In this paper, we report that by introducing new surface functional groups into the α-hemolysin pore, facilitated unzipping of double-stranded DNA through the channel could be achieved. Since the mean residence time of the DNA events is dependent on the length of the duplex, and also varies with the nucleotide base composition, the modified protein pore approach offers the potential for rapid double-stranded DNA analysis, including sequencing.     

Modulation of the translocation of peptides through nanopores by the application of an AC electric field

The interaction of two peptides with the α-hemolysin pore was studied in the presence of a MHz AC field. For an α-helical peptide the proportion of bumping events increased with increasing AC field whereas for a linear peptide with no dipole moment only small changes in the event profiles were observed.     

Application of photoactive yellow protein as a photoresponsive module for controlling hemolytic activity of staphylococcal α-hemolysin

A chimeric protein (N-PYP-Hla), consisting of staphylococcal pore-forming toxin α-hemolysin (Hla) and photoactive yellow protein (PYP), exhibited photoresponsive hemolytic activities, where visible light irradiation gave rise to retardation of hemolysis at 25 °C.     

S-nitrosothiol chemistry at the single-molecule level

SNO patrol: S-Nitrosothiols (RSNO) are important molecules involved in cell signaling, which control physiological processes such as vasodilation and bronchodilation. By using the protein pore α-hemolysin as a nanoreactor, the biological chemistry of RSNO has been investigated at the single-molecule level.     

Tuning the cavity of cyclodextrins: altered sugar adaptors in protein pores

Cyclodextrins (CDs) have been widely used in host-guest molecular recognition because of their chiral and hydrophobic cavities. For example, β-cyclodextrin (βCD) lodged as a molecular adaptor in protein pores such as α-hemolysin (αHL) is used for stochastic sensing. Here, we have tuned the cavity and overall size of βCD by replacing a single oxygen atom in its ring skeleton by a disulfide unit in two different configurations to both expand our ability to detect analytes and understand the interactions of βCD with protein pores. The three-dimensional structures of the two stereoisomeric CDs have been determined by the combined application of NMR spectroscopy and molecular simulation and show distorted conformations as compared to natural βCD. The interactions of these synthetic βCD analogues with mutant αHL protein pores and guest molecules were studied by single-channel electrical recording. The dissociation rate constants for both disulfide CDs from the mutant pores show ～1000-fold increase as compared to those of unaltered βCD, but are ～10-fold lower than the dissociation rate constants for βCD from wild-type αHL. Both of the skeleton-modified CDs show altered selectivity toward guest molecules. Our approach expands the breadth in sensitivity and diversity of sensing with protein pores and suggests structural parameters useful for CD design, particularly in the creation of asymmetric cavities.     

Highly sensitive and selective DNA-based detection of mercury(II) with α-hemolysin nanopore

The duplex formation mediated by Hg(2+) in a properly designed ssDNA generates a stable hairpin structure, which greatly alters the translocation profile of the ssDNA through α-hemolysin nanopore. From the 2D-events contour plot, the presence of Hg(2+) can be confirmed in as little as 30 min at ～7 nM or higher. The sensor is highly selective to Hg(2+), without interference from other metal ions. It can be fabricated from readily available materials, without the processes of synthesis, purification, probe-making, and so forth. This sensing strategy opens new possibilities for detecting many types of analytes which have specific interactions with DNA molecules.     

Microcontact printing for creation of patterned lipid bilayers on tetraethylene glycol self-assembled monolayers

Supported lipid bilayers (SLBs) formed on many different substrates have been widely used in the study of lipid bilayers. However, most SLBs suffer from inhomogeneities due to interactions between the lipid bilayer and the substrate. In order to avoid this problem, we have used microcontact printing to create patterned SLBs on top of ethylene-glycol-terminated self-assembled monolayers (SAMs). Glycol-terminated SAMs have previously been shown to resist absorbance of biomolecules including lipid vesicles. In our system, patterned lipid bilayer regions are separated by lipid monolayers, which form over the patterned hexadecanethiol portions of the surface. Furthermore, we demonstrate that α-hemolysin, a large transmembrane protein, inserts preferentially into the lipid bilayer regions of the substrate.     

Recognizing the translocation signals of individual peptide-oligonucleotide conjugates using an α-hemolysin nanopore

Two peptide-oligonucleotide conjugates are studied using an α-hemolysin nanopore to investigate their structural properties at the single-molecule level.     

Mutational analysis of human profilin I reveals a second PI(4,5)-P2 binding site neighbouring the poly(L-proline) binding site

Background

Profilin is a small cytoskeletal protein which interacts with actin, proline-rich proteins and phosphatidylinositol 4,5-bisphosphate (PI(4,5)-P₂). Crystallography, NMR and mutagenesis of vertebrate profilins have revealed the amino acid residues that are responsible for the interactions with actin and poly(L-proline) peptides. Although Arg88 of human profilin I was shown to be involved in PI(4,5)-P₂-binding, it was suggested that carboxy terminal basic residues may be involved as well.

Results

Using site directed mutagenesis we have refined the PI(4,5)-P₂ binding site of human profilin I. For each mutant we assessed the stability and studied the interactions with actin, a proline-rich peptide and PI(4,5)-P₂ micelles. We identified at least two PI(4,5)-P₂-binding regions in human profilin I. As expected, one region comprises Arg88 and overlaps with the actin binding site. The second region involves Arg136 in the carboxy terminal helix and neighbours the poly(L-proline) binding site. In addition, we show that adding a small protein tag to the carboxy terminus of profilin strongly reduces binding to poly(L-proline), suggesting local conformational changes of the carboxy terminal α-helix may have dramatic effects on ligand binding.

Conclusions

The involvement of the two terminal α-helices of profilin in ligand binding imposes important structural constraints upon the functions of this region. Our data suggest a model in which the competitive interactions between PI(4,5)-P₂ and actin and PI(4,5)-P₂ and poly(L-proline) regulate profilin functions.     

Peptide–Column Interactions and Their Influence on Back Exchange Rates in Hydrogen/Deuterium Exchange-MS

Hydrogen/deuterium exchange (HDX) methods generate useful information on protein structure and dynamics, ideally at the individual residue level. Most MS-based HDX methods involve a rapid proteolytic digestion followed by LC/MS analysis, with exchange kinetics monitored at the peptide level. Localizing specific sites of HDX is usually restricted to a resolution the size of the host peptide because gas-phase processes can scramble deuterium throughout the peptide. Subtractive methods may improve resolution, where deuterium levels of overlapping and nested peptides are used in a subtractive manner to localize exchange to smaller segments. In this study, we explore the underlying assumption of the subtractive method, namely, that the measured back exchange kinetics of a given residue is independent of its host peptide. Using a series of deuterated peptides, we show that secondary structure can be partially retained under quenched conditions, and that interactions between peptides and reversed-phase LC columns may both accelerate and decelerate residue HDX, depending upon peptide sequence and length. Secondary structure is induced through column interactions in peptides with a solution-phase propensity for structure, which has the effect of slowing HDX rates relative to predicted random coil values. Conversely, column interactions can orient random-coil peptide conformers to accelerate HDX, the degree to which correlates with peptide charge in solution, and which can be reversed by using stronger ion pairing reagents. The dependency of these effects on sequence and length suggest that subtractive methods for improving structural resolution in HDX-MS will not offer a straightforward solution for increasing exchange site resolution.

Structural basis for the activation of platelet integrin αIIbβ3 by calcium- and integrin-binding protein 1

Calcium and integrin binding protein 1 (CIB1) is a specific binding partner for the cytoplasmic domain of the αIIb subunit of the highly abundant platelet integrin αIIbβ3. This protein has been suggested to be involved in the regulation of the activation of αIIbβ3, a process leading to platelet aggregation and blood coagulation. In this work, the solution structure of the deuterated Ca(2+)-CIB1 protein complexed with an αIIb peptide was first determined through modern RDC-based NMR methods. Next, we generated a complex structure for CIB1 and the αIIb domain (Ca(2+)-CIB1/αIIb) using the program Haddock, which is based on experimental restraints obtained for the protein-peptide interface from cross-saturation NMR experiments. In this data-driven complex structure, the N-terminal α-helix of the cytoplasmic domain of αIIb is buried in the hydrophobic pocket of the C-lobe of Ca(2+)-CIB1. The C-terminal acidic tail of αIIb remains unstructured and likely interacts with several positively charged residues in the N-lobe of Ca(2+)-CIB1. A potential molecular mechanism for the CIB1-mediated activation of the platelet integrin could be proposed on the basis of the model structure of this protein complex. Another feature of this work is that, in the NMR cross-saturation experiments, we applied the selective radio frequency irradiation to the smaller binding partner (the αIIb peptide), and successfully detected the binding interface on the larger binding partner Ca(2+)-CIB1 through its selectively protonated methyl groups. This 'reverse' methodology has a broad potential to be employed to many other complexes where synthetic peptides and a suitably isotope-labeled medium- to large-sized protein are used to study protein-protein interactions.     

Formation of individual protein channels in lipid bilayers suspended in nanopores

Free-standing lipid bilayers are formed in regularly arranged nanopores of 200, 400 and 800 nm in a 300 nm thin hydrophobic silicon nitride membrane separating two fluid compartments. The extraordinary stability of the lipid bilayers allows us to monitor channel formation of the model peptide melittin and α-hemolysin from Staphylococcus aureus using electrochemical impedance spectroscopy and chronoamperometry. We observed that melittin channel formation is voltage-dependent and transient, whereas transmembrane heptameric α-hemolysin channels in nano-BLMs persist for hours. The onset of α-hemolysin-mediated conduction depends on the applied protein concentration and strongly on the diameter of the nanopores. Heptameric channel formation from adsorbed α-hemolysin monomers needs more time in bilayers suspended in 200 nm pores compared to bilayers in pores of 400 and 800 nm diameters. Diffusion of sodium ions across α-hemolysin channels present in a sufficiently high number in the bilayers was quantitatively and specifically determined using ion selective electrodes. The results demonstrate that relatively small variations of nano-dimensions have a tremendous effect on observable dynamic biomolecular processes. Such nanopore chips are potentially useful as supports for stable lipid bilayers to establish functional assays of membrane proteins needed in basic research and drug discovery.     

A Diazirine-Modified Membrane Lipid to Study Peptide/Lipid Interactions – Chances and Challenges

Although incorporation of photo-activatable lipids into membranes potentially opens up novel avenues for investigating interactions with proteins, the question of whether diazirine-modified lipids are suitable for such studies, remains under debate. Focusing on the potential for studying lipid/peptide interactions by cross-linking mass spectrometry (XL-MS), we developed a diazirine-modified lipid (DiazPC), and examined its behaviour in membranes incorporating the model α-helical peptide LAVA20. We observed an unexpected backfolding of the diazirine-containing stearoyl chain of the lipid. This surprising behaviour challenges the potential application of DiazPC for future XL-MS studies of peptide and protein/lipid interactions. The observations made for DiazPC most likely represent a general phenomenon for any type of membrane lipids with a polar moiety incorporated into the alkyl chain. Our finding is therefore of importance for future protein/lipid interaction studies relying on modified lipid probes.     

The balance between side-chain and backbone-driven association in folding of the α-helical influenza A transmembrane peptide

The correct balance between attractive, repulsive and peptide hydrogen bonding interactions must be attained for proteins to fold correctly. To investigate these important contributors, we sought a comparison of the folding between two 25-residues peptides, the influenza A M2 protein transmembrane domain (M2TM) and the 25-Ala (Ala₂₅). M2TM forms a stable α-helix as is shown by circular dichroism (CD) experiments. Molecular dynamics (MD) simulations with adaptive tempering show that M2TM monomer is more dynamic in nature and quickly interconverts between an ensemble of various α-helical structures, and less frequently turns and coils, compared to one α-helix for Ala₂₅. DFT calculations suggest that folding from the extended structure to the α-helical structure is favored for M2TM compared with Ala₂₅. This is due to CH⋯O attractive interactions which favor folding to the M2TM α-helix, and cannot be described accurately with a force field. Using natural bond orbital (NBO) analysis and quantum theory atoms in molecules (QTAIM) calculations, 26 CH⋯O interactions and 22 NH⋯O hydrogen bonds are calculated for M2TM. The calculations show that CH⋯O hydrogen bonds, although individually weaker, have a cumulative effect that cannot be ignored and may contribute as much as half of the total hydrogen bonding energy, when compared to NH⋯O, to the stabilization of the α-helix in M2TM. Further, a strengthening of NH⋯O hydrogen bonding interactions is calculated for M2TM compared to Ala₂₅. Additionally, these weak CH⋯O interactions can dissociate and associate easily leading to the ensemble of folded structures for M2TM observed in folding MD simulations.     

Negatively charged phospholipids trigger the interaction of a bacterial Tat substrate precursor protein with lipid monolayers

Folded proteins can be translocated across biological membranes via the Tat machinery. It has been shown in vitro that these Tat substrates can interact with membranes prior to translocation. Here we report a monolayer and infrared reflection-absorption spectroscopic (IRRAS) study of the initial states of this membrane interaction, the binding to a lipid monolayer at the air/water interface serving as a model for half of a biological membrane. Using the model Tat substrate HiPIP (high potential iron-sulfur protein) from Allochromatium vinosum, we found that the precursor preferentially interacts with monolayers of negatively charged phospholipids. The signal peptide is essential for the interaction of the precursor protein with the monolayer because the mature HiPIP protein showed no interaction with the lipid monolayer. However, the individual signal peptide interacted differently with the monolayer compared to the complete precursor protein. IRRA spectroscopy indicated that the individual signal peptide forms mainly aggregated β-sheet structures. This β-sheet formation did not occur for the signal peptide when being part of the full length precursor. In this case it adopted an α-helical structure upon membrane insertion. The importance of the signal peptide and the mature domain for the membrane interaction is discussed in terms of current ideas of Tat substrate-membrane interactions.     

Nanopore detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine in immobilized single-stranded DNA via adduct formation to the DNA damage site

The ability to detect DNA damage within the context of the surrounding sequence is an important goal in medical diagnosis and therapies, but there are no satisfactory methods available to detect a damaged base while providing sequence information. One of the most common base lesions is 8-oxo-7,8-dihydroguanine, which occurs during oxidation of guanine. In the work presented here, we demonstrate the detection of a single oxidative damage site using ion channel nanopore methods employing α-hemolysin. Hydantoin lesions produced from further oxidation of 8-oxo-7,8-dihydroguanine, as well as spirocyclic adducts produced from covalently attaching a primary amine to the spiroiminodihydantoin lesion, were detected by tethering the damaged DNA to streptavidin via a biotin linkage and capturing the DNA inside an α-hemolysin ion channel. Spirocyclic adducts, in both homo- and heteropolymer background single-stranded DNA sequences, produced current blockage levels differing by almost 10% from those of native base current blockage levels. These preliminary studies show the applicability of ion channel recordings not only for DNA sequencing, which has recently received much attention, but also for detecting DNA damage, which will be an important component to any sequencing efforts.     

An agent-based system to discover protein-protein interactions, identify protein complexes and proteins with multiple peptide mass fingerprints

Proteins "work together" by actually binding to form multicomponent complexes that carry out specific functions. Proteomic analyses based on the mass spectrum are now key methods to determine the components in protein complexes. The protein-protein interaction or functional association may be known to exist among the extracted protein spots while analyzing the proteins on the 2D gel. In this study, we develop an agent-based system, namely AgentMultiProtIdent, which integrated two protein identification tools and a variety of databases storing relations among proteins and used to discover protein-protein interactions and protein functional associations, and identify protein complexes and proteins with multiple peptide mass fingerprints as input. The system takes Multiple Peptide Mass Fingerprints (PMFs) as a whole in the protein complex or protein identification. With the relations among proteins, it may greatly improve the accuracy of identification of protein complexes. Also, possible relationship of the multiple peptide mass fingerprints, such as ontology relation, can be discovered by our system, especially in the identification of protein complexes. The agent-based system is now available on the Web at http://dbms104.csie.ncu.edu.tw/ approximately protein/NEW2/.     

Dynamics of unfolded protein transport through an aerolysin pore

Protein export is an essential mechanism in living cells and exported proteins are usually translocated through a protein-conducting channel in an unfolded state. Here we analyze, by electrical detection, the entry and transport of unfolded proteins, at the single molecule level, with different stabilities through an aerolysin pore, as a function of the applied voltage and protein concentration. The frequency of ionic current blockades varies exponentially as a function of the applied voltage and linearly as a function of protein concentration. The transport time of unfolded proteins decreases exponentially when the applied voltage increases. We prove that the ionic current blockade duration of a double-sized protein is longer than that assessed for a single protein supporting the transport phenomenon. Our results fit with the theory of confined polyelectrolyte and with some experimental results about DNA or synthetic polyelectrolyte translocation through protein channels as a function of applied voltage. We discuss the potential of the aerolysin nanopore as a tool for protein folding studies as it has already been done for α-hemolysin.     

Enhanced translocation of poly(dt)45 through an α-hemolysin nanopore by binding with antibody

The translocation time of poly(dT)(45) through an α-hemolysin pore was reduced in the presence of a DNA-binding Fab fragment. The Fab acts as a rudder to steer the DNA into the pore.     

Bicyclic stapled peptides based on p53 as dual inhibitors for the interactions of p53 with MDM2 and MDMX

In recent years, the strategy of inhibiting the interactions of p53 with murine double minute 2(MDM2)and murine double minute X(MDMX) has been proved to be a promising approach for tumor therapy.However, the poor proteolytical stability and low intracellular delivery efficiency of peptide inhibitors limit their clinical application. Here, we designed and synthesized the bicyclic stapled peptides based on p53 by combining all-hydrocarbon stapling and lactam stapling strategies. We demonstrated th...