Alexander Negoda

Insight into the selectivity and gating functions of Streptomyces lividans KcsA.

Streptomyces lividans KcsA is a 160-aa polypeptide that oligomerizes to form a tetrameric potassium channel. The three-dimensional structure of the polypeptides has been established, but the selectivity and gating functions of the channel remain unclear. It has been shown that the polypeptides copurify with two homopolymers, poly[(R)-3-hydroxybutyrate] (PHB) and inorganic polyphosphate (polyP), which have intrinsic capacities for cation selection and transport. PHB/polyP complexes are highly selective for divalent cations when pH is greater than the pK2 of polyP (≈6.8), but this preference is lost when pH is ≤pK2. It is postulated that KcsA polypeptides attenuate the divalent negative charge of the polyP end unit at physiological pH by strategic positioning of two C-terminal arginines. Here we mutate one or both of the C-terminal arginines and observe the effects on channel selectivity in planar lipid bilayers. We find that channels formed by KcsA polypeptides that retain a single C-terminal arginine remain highly selective for K+ over Mg2+, independent of medium pH; however, channels formed by KcsA polypeptides in which both C-terminal arginines have been replaced with neutral residues are selective for Mg2+ when pH is >7 and for K+ when pH is <7. Channel gating may be triggered by changes in the balance between the K+ polyP− binding energy, the membrane potential, and the gradient force. The results reveal the importance of the C-terminal arginines to K+ selectivity and argue for a supramolecular structure for KcsA in which the host polypeptides modify the cation preference of a guest PHB/polyP complex.

Engineered nanomaterial interactions with bilayer lipid membranes: screening platforms to assess nanoparticle toxicity

Engineered nanomaterials (ENMs) have attractive functional properties and are increasingly being used in commercial products. However, ENMs present health risks that are poorly understood and difficult to assess. Because ENMs must interface with cell membranes to cause biological effects, improved methods are needed to measure ENM-biomembrane interactions. The goals of this paper are to review the current status of methods to characterise interactions between ENMs and bilayer lipid membranes that mimic cell membranes, and to present example applications of the methods relevant to nanotoxicology. Four approaches are discussed: electrochemical methods that measure ENM-induced ion leakage through lipid bilayers, optical methods that measure dye leakage from liposomes, partitioning methods that measure ENM distribution coefficients between aqueous solution and immobilised lipid bilayers, and theoretical models capable of predicting fundamental molecular interactions between ENMs and biomembranes. For each approach, current literature is summarised, recent results are given, and future prospects are analysed, including the potential to be used in a high-throughput mode. The relative advantages of the various approaches are discussed, along with their synergistic potential to provide multi-dimensional characterisation of ENM-biomembrane interactions for robust health risk assessment algorithms.

Resolving the native conformation of Escherichia coli OmpA

The native conformation of the 325‐residue outer membrane protein A (OmpA) of Escherichia coli has been a matter of contention. A narrow‐pore, two‐domain structure has vied with a large‐pore, single‐domain structure. Our recent studies show that Ser163 and Ser167 of the N‐terminal domain (1–170) are modified in the cytoplasm by covalent attachment of oligo‐(R)‐3‐hydroxybutyrates (cOHBs), and further show that these modifications are essential for the N‐terminal domain to be incorporated into planar lipid bilayers as narrow pores (∼ 80 pS, 1 m KCl, 22 °C). Here, we examined the potential effect(s) of periplasmic modifications on pore structure by comparing OmpA isolated from outer membranes (M‐OmpA) with OmpA isolated from cytoplasmic inclusion bodies (I‐OmpA). Chemical and western blot analysis and 1H‐NMR showed that segment 264–325 in M‐OmpA, but not in I‐OmpA, is modified by cOHBs. Moreover, a disulfide bond is formed between Cys290 and Cys302 by the periplasmic enzyme DsbA. Planar lipid bilayer studies indicated that narrow pores formed by M‐OmpA undergo a temperature‐induced transition into stable large pores (∼ 450 pS, 1 m KCl, 22 °C) [energy of activation (Ea) = 33.2 kcal·mol−1], but this transition does not occur with I‐OmpA or with M‐OmpA that has been exposed to disulfide bond‐reducing agents. The results suggest that the narrow pore is a folding intermediate, and demonstrate the decisive roles of cOHB‐modification, disulfide bond formation and temperature in folding OmpA into its native large‐pore configuration.

Oligo-(R)-3-hydroxybutyrate modification of sorting signal enables pore formation by Escherichia coli OmpA.

The outer membrane protein A (OmpA) of Escherichia coli is a well-known model for protein targeting and protein folding. Wild-type OmpA, isolated either from cytoplasmic inclusion bodies or from outer membranes, forms narrow pores of approximately 80 pS in planar lipid bilayers at room temperature. The pores are well structured with narrow conductance range when OmpA is isolated using lithium dodecyl sulfate (LDS) or RapiGest surfactant but display irregular conductance when OmpA is isolated with urea or guanidine hydrochloride. Previous studies have shown that serine residues S163 and S167 of the sorting signal of OmpA (residues 163-169), i.e., the essential sequence for outer membrane incorporation, are covalently modified by oligomers of (R)-3-hydroxybutyrate (cOHB). Here we find that single-mutants S163 and S167 of OmpA, which still contain cOHB on one serine of the sorting signal, form narrow pores in planar lipid bilayers at room temperature with lower and more irregular conductance than wild-type OmpA, whereas double mutants S163:S167 and S163:V166 of OmpA, with no cOHB on the sorting signal, are unable to form stable pores in planar lipid bilayers. Our results indicate that modification of serines in the sorting signal of OmpA by cOHB in the cytoplasm enables OmpA to incorporate into lipid bilayers at room temperature as a narrow pore. They further suggest that cOHB modification may be an important factor in protein targeting and protein folding.

Role of polyphosphate in regulation of the Streptomyces lividans KcsA channel

We examine the hypotheses that the Streptomyces lividans potassium channel KcsA is gated at neutral pH by the electrochemical potential, and that its selectivity and conductance are governed at the cytoplasmic face by interactions between the KcsA polypeptides and a core molecule of inorganic polyphosphate (polyP). The four polypeptides of KcsA are postulated to surround the end unit of the polyP molecule with a collar of eight arginines, thereby modulating the negative charge of the polyP end unit and increasing its preference for binding monovalent cations. Here we show that KcsA channels can be activated in planar lipid bilayers at pH 7.4 by the chemical potential alone. Moreover, one or both of the C-terminal arginines are replaced with residues of progressively lower basicity-lysine, histidine, valine, asparagine-and the effects of these mutations on conductance and selectivity for K(+) over Mg(2+) is tested in planar bilayers as a function of Mg(2+) concentration and pH. As the basicity of the C-terminal residues decreases, Mg(2+) block increases, and Mg(2+) becomes permeant when medium pH is greater than the pI of the C-terminal residues. The results uphold the premise that polyP and the C-terminal arginines are decisive elements in KcsA channel regulation.

Polystyrene nanoparticle exposure induces ion-selective pores in lipid bilayers

A diverse range of molecular interactions can occur between engineered nanomaterials (ENM) and biomembranes, some of which could lead to toxic outcomes following human exposure to ENM. In this study, we adapted electrophysiology methods to investigate the ability of 20nm polystyrene nanoparticles (PNP) to induce pores in model bilayer lipid membranes (BLM) that mimic biomembranes. PNP charge was varied using PNP decorated with either positive (amidine) groups or negative (carboxyl) groups, and BLM charge was varied using dioleoyl phospholipids having cationic (ethylphosphocholine), zwitterionic (phosphocholine), or anionic (phosphatidic acid) headgroups. Both positive and negative PNP induced BLM pores for all lipid compositions studied, as evidenced by current spikes and integral conductance. Stable PNP-induced pores exhibited ion selectivity, with the highest selectivity for K(+) (PK/PCl~8.3) observed when both the PNP and lipids were negatively charged, and the highest selectivity for Cl(-) (PK/PCl~0.2) observed when both the PNP and lipids were positively charged. This trend is consistent with the finding that selectivity for an ion in channel proteins is imparted by oppositely charged functional groups within the channels filter region. The PK/PCl value was unaffected by the voltage-ramp method, the pore conductance, or the side of the BLM to which the PNP were applied. These results demonstrate for the first time that PNP can induce ion-selective pores in BLM, and that the degree of ion selectivity is influenced synergistically by the charges of both the lipid headgroups and functional groups on the PNP.