Directed Evolution and Engineering of Gallium-Binding Phage Clones—A Preliminary Study

The phage surface display technology is a useful tool to screen and to extend the spectrum of metal-binding protein structures provided by nature. The directed evolution approach allows identifying specific peptide ligands for metals that are less abundant in the biosphere. Such peptides are attractive molecules in resource technology. For example, gallium-binding peptides could be applied to recover gallium from low concentrated industrial wastewater. In this study, we investigated the affinity and selectivity of five bacteriophage clones displaying different gallium-binding peptides towards gallium and arsenic in independent biosorption experiments. The displayed peptides were highly selective towards Ga3+ whereby long linear peptides showed a lower affinity and specificity than those with a more rigid structure. Cysteine scanning was performed to determine the relationship between secondary peptide structure and gallium sorption. By site-directed mutagenesis, the amino acids of a preselected peptide sequence are systematically replaced by cysteines. The resulting disulphide bridge considerably reduces the flexibility of linear peptides. Subsequent biosorption experiments carried out with the mutants obtained from cysteine scanning demonstrated, depending on the position of the cysteines in the peptide, either a considerable increase in the affinity of gallium compared to arsenic or an increase in the affinity for arsenic compared to gallium. This study shows the impressive effect on peptide–target interaction based on peptide structure and amino acid position and composition via the newly established systematic cysteine scanning approach.

Organization of ATP-gated P2X1 receptor intracellular termini in apo and desensitized states

The human P2X1 receptor (hP2X1R) is a trimeric ligand-gated ion channel opened by extracellular ATP. The intracellular amino and carboxyl termini play significant roles in determining the time-course and regulation of channel gating—for example, the C terminus regulates recovery from the desensitized state following agonist washout. This suggests that the intracellular regions of the channel have distinct structural features. Studies on the hP2X3R have shown that the intracellular regions associate to form a cytoplasmic cap in the open state of the channel. However, intracellular features could not be resolved in the agonist-free apo and ATP-bound desensitized structures. Here we investigate the organization of the intracellular regions of hP2X1R in the apo and ATP-bound desensitized states following expression in HEK293 cells. We couple cysteine scanning mutagenesis of residues R25-G30 and H355-R360 with the use of bi-functional cysteine reactive cross-linking compounds of different lengths (MTS-2-MTS, BMB, and BM(PEG)2), which we use as molecular calipers. If two cysteine residues come into close proximity, we predict they will be cross-linked and result in ∼66% of the receptor subunits running on a Western blot as dimers. In the control construct (C349A) that removed the free cysteine C349, and some cysteine-containing mutants, cross-linker treatment does not result in dimerization. However, we detect efficient dimerization for R25C, G30C, P358C, K359C, and R360C. This selective pattern indicates that there is structural organization to these regions in the apo and desensitized states in a native membrane environment. The existence of such precap (apo) and postcap (desensitized) organization of the intracellular domains would facilitate efficient gating of the channel.

PrPSc-induced conformational changes and strain-specific structures of PrPSc revealed by Disulfide-crosslink scanning

ABSTRACTThere exist many phenotypically-varied prion strains, like viruses, despite the absence of conventional genetic material which codes the phenotypic information. As prion is composed solely of the pathological isoform (PrPSc) of prion protein (PrP), the strain-specific traits are hypothesized to be enciphered in the structural details of PrPSc. Identification of the structures of PrPSc is therefore vital for the understanding of prion biology, though they remain unidentified due to the incompatibility of PrPSc with conventional high-resolution structural analyses. Based on our previous hypothesis that the region between the first and the second α-helix (H1∼H2) and the distal region of the third helix (Ctrm) of the cellular isoform of PrP (PrPC) have important roles for efficient interactions with PrPSc, we created series of mutant PrPs with two cysteine substitutions (C;C-PrP) which were systematically designed to form an intramolecular disulfide crosslink between H1∼H2 and Ctrm and assessed their conformational changes by prions: Specifically, a cysteine substitution in H1∼H2 from 165 to 169 was combined with cysteine-scanning along Ctrm from 220 to 229. C;C-PrPs with the crosslinks were expressed normally with the similar glycosylation patterns and subcellular localization as the wild-type PrP albeit with varied expression levels. Interestingly, some of the C;C-PrPs converted to the protease-resistant isoforms in the N2a cells persistently infected with 22L prion strain, whereas the same mutants did not convert in the cells infected with another prion strain Fukuoka1, indicating that local structures of PrPSc in these regions vary among prion strains and contribute to prion-strain diversity. Moreover, patterns of the crosslinks of the convertible C;C-PrPs implied drastic changes in positional relations of H1∼H2 and Ctrm in the PrPSc-induced conformational changes by 22L prion. Thus, disulfide-crosslink scanning is a useful approach for investigation of strain-specific structures of PrPSc, and would be applicable to other types of amyloids as well.

Glutathione release through connexin hemichannels: Implications for chemical modification of pores permeable to large molecules

Cysteine-scanning mutagenesis combined with thiol reagent modification is a powerful method with which to define the pore-lining elements of channels and the changes in structure that accompany channel gating. Using the Xenopus laevis oocyte expression system and two-electrode voltage clamp, we performed cysteine-scanning mutagenesis of several pore-lining residues of connexin 26 (Cx26) hemichannels, followed by chemical modification using a methanethiosulfonate (MTS) reagent, to help identify the position of the gate. Unexpectedly, we observed that the effect of MTS modification on the currents was reversed within minutes of washout. Such a reversal should not occur unless reducing agents, which can break the disulfide thiol–MTS linkage, have access to the site of modification. Given the permeability to large metabolites of connexin channels, we tested whether cytosolic glutathione (GSH), the primary cell reducing agent, was reaching the modified sites through the connexin pore. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine decreased the cytosolic GSH concentration in Xenopus oocytes and reduced reversibility of MTS modification, as did acute treatment with tert-butyl hydroperoxide, which oxidizes GSH. Cysteine modification based on thioether linkages (e.g., maleimides) cannot be reversed by reducing agents and did not reverse with washout. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay, we confirmed that homomeric Cx26 and Cx32 and heteromeric Cx26/Cx32 are permeable to GSH and other endogenous reductants. These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications. This potential for reversibility of thiol modification applies to on-cell accessibility studies of connexin channels and other channels that are permeable to large molecules, such as pannexin, CALHM, and VRAC.