Bioorthogonal catalytic patch.

Bioorthogonal catalysis mediated by transition metals has inspired a new subfield of artificial chemistry complementary to enzymatic reactions, enabling the selective labelling of biomolecules or in situ synthesis of bioactive agents via non-natural processes. However, the effective deployment of bioorthogonal catalysis in vivo remains challenging, mired by the safety concerns of metal toxicity or complicated procedures to administer catalysts. Here, we describe a bioorthogonal catalytic device comprising a microneedle array patch integrated with Pd nanoparticles deposited on TiO2 nanosheets. This device is robust and removable, and can mediate the local conversion of caged substrates into their active states in high-level living systems. In particular, we show that such a patch can promote the activation of a prodrug at subcutaneous tumour sites, restoring its parent drug's therapeutic anticancer properties. This in situ applied device potentiates local treatment efficacy and eliminates off-target prodrug activation and dose-dependent side effects in healthy organs or distant tissues.

Membrane Topology of an Ion Channel Detected by Solid-State Nuclear Magnetic Resonance and Paramagnetic Effects.

Ion channels are often targeted by toxins or other ligands to modify their channel activities and alter ion conductance. Interactions between toxins and ion channels could result in changes in membrane insertion depth for residues close to the binding site. Paramagnetic solid-state nuclear magnetic resonance (SSNMR) has shown great potential in providing structural information on membrane samples. We used KcsA as a model ion channel to investigate how the paramagnetic effects of Mn2+ and Dy3+ ions with headgroup-modified chelator lipids would influence the SSNMR signals of membrane proteins in proteoliposomes. Spectral comparisons have shown significant changes of peak intensities for the residues in the loop or terminal regions due to paramagnetic effects corresponding to the close proximity to the membrane surface. Hence, these results demonstrate that paramagnetic SSNMR can be used to detect surface residues based on the topology and membrane insertion properties for integral membrane proteins.

Observation of the Imidazole-Imidazolium Hydrogen Bonds Responsible for Selective Proton Conductance in the Influenza A M2 Channel.

The integral membrane M2 protein is a 97-residue membrane protein that assembles as a tetramer to conduct protons at a slow rate (102-103/s) when activated by low pH. The proton conductance mechanism has been extensively debated in the literature, but it is accepted that the proton conductance is facilitated by hydrogen bonds involving the His37 residues. However, the hydrogen bonding partnership remains unresolved. Here, we report on the measurement of 15N-15N J-couplings of 15N His37-labeled full length M2 (M2FL) protein from Influenza A virus embedded in synthetic liquid crystalline lipid bilayers using two-dimensional J-resolved NMR spectroscopy. We experimentally observed the hydrogen-bond mediated J-couplings between Nδ1 and Nε2 of adjacent His37 imidazole rings, providing direct evidence for the existence of various imidazolium-imidazole hydrogen-bonding geometries in the histidine tetrad at low pH, thus validating the proton conduction mechanism in the M2FL protein by which the proton is transferred through the breaking and reforming of the hydrogen bonds between pairs of His37 residues.

Evolutionary and genotypic analyses of global porcine epidemic diarrhea virus strains.

Porcine epidemic diarrhea virus (PEDV), which re-emerged in China in October 2010, has spread rapidly worldwide. Detailed analyses of the complete genomes of different PEDV strains are essential to understand the relationships among re-emerging and historic strains worldwide. Here, we analysed the complete genomes of 409 strains from different countries, which were classified into five subgroup strains (i.e., GI-a, GI-b, GII-a, GII-b, and GII-c). Phylogenetic study of different genes in the PEDV strains revealed that the newly discovered subgroup GII-c exhibited inconsistent topologies between the spike gene and other genes. Furthermore, recombination analysis indicated that GII-c viruses evolved from a recombinant virus that acquired the 5' part of the spike gene from the GI-a subgroup and the remaining genomic regions from the GII-a subgroup. Molecular clock analysis showed that divergence of the GII-c subgroup spike gene occurred in April 2010, suggesting that the subgroup originated from recombination events before the PEDV re-emergence outbreaks. Interestingly, Ascaris suum, a large roundworm occurring in pigs, was found to be an unusual PEDV host, providing potential support for cross-host transmission. This study has significant implications for understanding ongoing global PEDV outbreaks and will guide future efforts to develop effective preventative measures against PEDV.

Beyond Structural Biology to Functional Biology: Solid-State NMR Experiments and Strategies for Understanding the M2 Proton Channel Conductance.

In terms of structural biology, solid-state NMR experiments and strategies have been well established for resonance assignments, leading to the determination of three-dimensional structures of insoluble membrane proteins in their native-like environment. It is also known that NMR has the unique capabilities to characterize structure-function relationships of membrane-bound biological systems beyond structural biology. Here, we report on solid-state NMR experiments and strategies for extracting functional activities on a sub-millisecond time scale. Specifically, we use the His37-labeled full length M2 (M2FL) protein of the Influenza A virus embedded in synthetic lipid bilayers as an example to characterize the proton conduction mechanism and kinetics. The integral membrane M2 protein assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH and is essential for the viral lifecycle. Our results present convincing evidence for the formation of imidazolium-imidazole hydrogen bonds in the His37 tetrad at low pH and that these hydrogen bonds have a low barrier that facilitates the proton conduction mechanism in the M2FL protein. Moreover, it has been possible to measure hydronium ion exchange between water and the protons in the His37 NH bonds based on chemical exchange spectroscopy with minimized spin diffusion. The results identify an exchange rate constant of ∼4000 s-1 for pH 5.8 at -10 °C.

Probing Hydronium Ion Histidine NH Exchange Rate Constants in the M2 Channel via Indirect Observation of Dipolar-Dephased 15N Signals in Magic-Angle-Spinning NMR.

Water-protein chemical exchange in membrane-bound proteins is an important parameter for understanding how proteins interact with their aqueous environment, but has been difficult to observe in membrane-bound biological systems. Here, we demonstrate the feasibility of probing specific water-protein chemical exchange in membrane-bound proteins in solid-state MAS NMR. By spin-locking the 1H magnetization along the magic angle, the 1H spin diffusion is suppressed such that a water-protein chemical exchange process can be monitored indirectly by dipolar-dephased 15N signals through polarization transfer from 1H. In the example of the Influenza A full length M2 protein, the buildup of dipolar-dephased 15N signals from the tetrad of His37 side chains have been observed as a function of spin-lock time. This confirms that hydronium ions are in exchange with protons in the His37 NH bonds at the heart of the M2 proton conduction mechanism, with an exchange rate constant of ∼1750 s-1 for pH 6.2 at -10 °C.

Dynamic Short Hydrogen Bonds in Histidine Tetrad of Full-Length M2 Proton Channel Reveal Tetrameric Structural Heterogeneity and Functional Mechanism.

The tetrameric M2 protein from influenza A conducts protons into the virus upon acid activation of its His37 tetrad and is a proven drug target. Here, in studies of full-length M2 protein solubilized in native-like liquid-crystalline lipid bilayers, a pH titration monitored by solid-state nuclear magnetic resonance revealed a clustering of the first three His37 pKas (6.3, 6.3, and 5.5). When the +2 state of the tetrad accepts a third proton from the externally exposed portion of the channel pore and releases a proton to the internally exposed pore, successful proton conductance is achieved, but more frequently the tetrad accepts and returns the proton to the externally exposed pore, resulting in a futile cycle. Both dynamics and conformational heterogeneity of the His37 tetrad featuring short hydrogen bonds between imidazolium-imidazole pairs are characterized, and the heterogeneity appears to reflect oligomeric helix packing and the extent of transmembrane helical bending around Gly34.

Differentiation of histidine tautomeric states using (15)N selectively filtered (13)C solid-state NMR spectroscopy.

The histidine imidazole ring in proteins usually contains a mixture of three possible tautomeric states (two neutral - τ and π states and a charged state) at physiological pHs. Differentiating the tautomeric states is critical for understanding how the histidine residue participates in many structurally and functionally important proteins. In this work, one dimensional (15)N selectively filtered (13)C solid-state NMR spectroscopy is proposed to differentiate histidine tautomeric states and to identify all (13)C resonances of the individual imidazole rings in a mixture of tautomeric states. When (15)N selective 180° pulses are applied to the protonated or non-protonated nitrogen region, the (13)C sites that are bonded to the non-protonated or protonated nitrogen sites can be identified, respectively. A sample of (13)C, (15)N labeled histidine powder lyophilized from a solution at pH 6.3 has been used to illustrate the usefulness of this scheme by uniquely assigning resonances of the neutral τ and charged states from the mixture.

Solid state NMR and protein-protein interactions in membranes.

Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high-resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water-soluble proteins and other membrane proteins.

Identifying inter-residue resonances in crowded 2D (13)C- (13)C chemical shift correlation spectra of membrane proteins by solid-state MAS NMR difference spectroscopy.

The feasibility of using difference spectroscopy, i.e. subtraction of two correlation spectra at different mixing times, for substantially enhanced resolution in crowded two-dimensional (13)C-(13)C chemical shift correlation spectra is presented. With the analyses of (13)C-(13)C spin diffusion in simple spin systems, difference spectroscopy is proposed to partially separate the spin diffusion resonances of relatively short intra-residue distances from the longer inter-residue distances, leading to a better identification of the inter-residue resonances. Here solid-state magic-angle-spinning NMR spectra of the full length M2 protein embedded in synthetic lipid bilayers have been used to illustrate the resolution enhancement in the difference spectra. The integral membrane M2 protein of Influenza A virus assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH and is essential for the viral lifecycle. Based on known amino acid resonance assignments from amino acid specific labeled samples of truncated M2 sequences or from time-consuming 3D experiments of uniformly labeled samples, some inter-residue resonances of the full length M2 protein can be identified in the difference spectra of uniformly (13)C labeled protein that are consistent with the high resolution structure of the M2 (22-62) protein (Sharma et al., Science 330(6003):509-512, 2010).

Dual-band selective double cross polarization for heteronuclear polarization transfer between dilute spins in solid-state MAS NMR.

A sinusoidal modulation scheme is described for selective heteronuclear polarization transfer between two dilute spins in double cross polarization magic-angle-spinning nuclear magnetic resonance spectroscopy. During the second N→C cross polarization, the (13)C RF amplitude is modulated sinusoidally while the (15)N RF amplitude is tangent. This modulation induces an effective spin-lock field in two selective frequency bands in either side of the (13)C RF carrier frequency, allowing for simultaneous polarization transfers from (15)N to (13)C in those two selective frequency bands. It is shown by experiments and simulations that this sinusoidal modulation allows one to selectively polarize from (15)N to its covalently bonded (13)Cα and (13)C' carbons in neighboring peptide planes simultaneously, which is useful for establishing the backbone connectivity between two sequential residues in protein structural elucidation. The selectivity and efficiency were experimentally demonstrated on a uniformly (13)C,(15)N-labeled ß1 immunoglobulin binding domain of protein G (GB1).