Radu I. Stefureac

Nanopore Analysis of the Folding of Zinc Fingers

In classical biochemistry, denatured or unfolded proteins were considered to be non-functional or inactive. However, recent progress in proteomics and genomics research suggests that as many as 30% of eukaryotic proteins are unfolded or contain significant disordered regions. (These proteins are also known as natively unfolded and intrinsically disordered). Many such proteins become folded upon interaction with DNA, other proteins, or metal ions and have also been implicated in disease pathogenesis. Examples include p53 and cancer, prion proteins and mad-cow disease, and a-synuclein and Parkinson’s disease. Needless to say, disordered proteins have proven difficult to study by conventional techniques, such as X-ray crystallography or NMR, because multiple conformations coexist. Single-molecule techniques may be used to overcome this problem as, by necessity, they examine a single conformation. Here, we demonstrate a simple model for conformational studies of protein folding involving the interaction of metal ions with Zn-finger peptides investigated by nanopore analysis. Recently, there have been several reports on the use of nanopores to investigate short peptides and small proteins, as well as antibody/protein and DNA/ protein complexes. The biological pore formed by a-hemolysin has proved to be very useful for this work since it self-assembles into a lipid membrane, yielding a channel with an internal diameter of about 1.5 nm. Under a constant voltage of 100mV in a buffer of 1 M KCl there is a constant current of about 100 pA, which is readily measured by patchclamp techniques. When a molecule approaches or enters the pore it causes a blockade current (I) for a particular time (T). The value of these parameters can be related to the charge, structure, and state of complexation of the molecule. If a protein is larger than the size of the pore then it may bump into the pore and then diffuse away which, in general, is characterized by a small value for I. Alternatively, the protein may unfold and translocate the pore, giving rise to a large I. Thus this technique can be used to study the state of folding of proteins and peptides at the single-molecule level.

Nanopore analysis of the interaction of metal ions with prion proteins and peptides.

Nanopore analysis can be used to study conformational changes in individual peptide or protein molecules. Under an applied voltage there is a change in the event parameters of blockade current or time when a molecule bumps into or translocates through the pore. If a molecule undergoes a conformational change upon binding a ligand or metal ion the event parameters will be altered. The objective of this research was to demonstrate that the conformation of the prion protein (PrP) and prion peptides can be modulated by binding divalent metal ions. Peptides from the octarepeat region (Octa2, (PHGGGWGQ)2 and Octa 4, (PHGGGWGQ)4), residues 106-126 (PrP106-126), and the full-length Bovine recombinant prion (BrecPrP) were studied with an alpha-hemolysin pore. Octa2 readily translocated the pore but significant bumping events occurred on addition of Cu(II) and to a lesser extent Zn(II), demonstrating that complex formation was occurring with concomitant conformational changes. The binding of Cu(II) to Octa4 was more pronounced and at high concentrations only a small proportion of the complex could translocate. Addition of Zn(II) also caused significant changes to the event parameters but Mg(II) and Mn(II) were inert. Addition of Cu(II) to PrP106-126 caused the formation of a very tight complex, which could not translocate the pore. Small changes were observed with Zn(II), but not with Mg(II) or Mn(II). Analysis of BrecPrP showed that about 37% were translocation events, but on addition of Cu(II) or Zn(II) these disappeared and only bumping events were recorded. Suprisingly, addition of Mn(II) caused an increase in translocation events to about 64%. Thus, conformational changes to prions upon binding metal ions are readily observed by nanopore analysis.

Nanopore analysis of tethered peptides

Peptides of 12 amino acids were tethered via a terminal cysteine to mono‐, di‐, tri‐, and tetrabromomethyl‐substituted benzene to produce bundles of one to four peptide strands (CY12‐T1 to CY12‐T4, respectively). The interaction of the bundles with the α‐hemolysin pore was assessed by measuring the blockade currents (I) and times (T) at an applied potential of − 50, − 100, and − 150 mV. Three types of events could be distinguished: bumping events, with small I and short T where the molecule transiently interacts with the pore before diffusing away; translocation events, where the molecule threads through the pore with large I and the value of T decreases with increasing voltage; and intercalation events, where the molecule transiently enters the pore but does not translocate with large I and the value of T increases with increasing voltage. CY12‐T1 and CY12‐T2 gave only bumping and translocation events; CY12‐T3 and CY12‐T4 also gave intercalation events, some of which were of very long duration. The results suggest that three uncoiled peptide strands cannot simultaneously thread through the α‐hemolysin pore and that proteins must completely unfold in order to translocate. Copyright

Effect of charge, topology and orientation of the electric field on the interaction of peptides with the α-hemolysin pore

Nanopore analysis is an emerging technique of structural biology which employs nanopores, such as the α‐hemolysin pore, as a biosensor. A voltage applied across a membrane containing a nanopore generates a current, which is partially blocked when a molecule interacts with the pore. The magnitude (I) and the duration (T) of the current blockade provide an event signature for that molecule. Two peptides, CY12(+)T1 and CY12(−)T1 with net charges + 2 and − 2, respectively, were analysed using different applied voltages and all four possible orientations of the electrodes and pore. The four orientations were vestibule downstream (VD), vestibule upstream (VU), stem downstream (SD) and stem upstream (SU) where vestibule and stem refer to the side of the pore on which the peptide was placed and downstream and upstream refer to the application of a positive or negative electrophoretic force, respectively. For CY12(+)T1, the effect of voltage on the event duration was consistent with translocation in the VD and SD configurations, but only intercalation events were observed in the VU and SU configurations. For CY12(−)T1, translocations were only observed in the VD and VU configurations. The results are interpreted in terms of two energy barriers on either side of the lumen of the pore. The difference in height of the barriers determines the preferred direction of exit. Electroosmotic flow and current rectification due to the pore as well as the dipole moment and charge of the peptide also play significant roles. Thus, factors other than simple electrophoresis are important for determining the interaction of small peptides with the pore. Copyright

Evidence that small proteins translocate through silicon nitride pores in a folded conformation

The interaction of three proteins (histidine-containing phosphocarrier protein, HPr, calmodulin, CaM, and maltose binding protein, MBP) with synthetic silicon nitride (SiN(x)) membranes has been studied. The proteins which have a net negative charge were electrophoretically driven into pores of 7 and 5 nm diameter with a nominal length of 15 nm. The % blockade current and event duration were measured at three different voltages. For a translocation event it was expected that the % block would be constant with voltage whilst the event duration would decrease with increasing voltage. On the basis of these criteria, we deduce that MBP whose largest dimension is 6.5 nm does not translocate whereas up to 40% of CaM molecules can translocate the 7 nm pore as can a majority of HPr molecules, with some translocations being observed for the 5 nm pore. For translocation events the magnitude of the % blockade current is consistent with a folded conformation of the proteins surrounded by a hydration shell of 0.5-1.0 nm.

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.