A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis.

Halorhodopsin (HR) is a seven-transmembrane retinylidene protein from haloarchaea that is commonly known to function as a light-driven inward chloride pump. However, previous studies have indicated that despite the general characteristics that most HRs share, HRs from distinct species differ in many aspects. We present indium-tin-oxide-based photocurrent measurements that reveal a light-induced signal generated by proton release that is observed solely in NpHR via purified protein-based assays, demonstrating that indeed HRs are not all identical. We conducted mutagenesis studies on several conserved residues that are considered critical for chloride stability among HRs. Intriguingly, the photocurrent signals were eliminated after specific point mutations. We propose an NpHR light-driven, cytoplasmic-side proton circulation model to explain the unique light-induced photocurrent recorded in NpHR. Notably, the photocurrent and various photocycle intermediates were recorded simultaneously. This approach provides a high-resolution method for further investigations of the proton-assisted chloride translocation mechanism.

Insight into a single halobacterium using a dual-bacteriorhodopsin system with different functionally optimized pH ranges to cope with periplasmic pH changes associated with continuous light illumination.

The light-driven outward proton transporter assists energy production via an ATP synthase system best exemplified by the bacteriorhodopsin (BR) from Halobacterium salinarum, HsBR. As the only archaea able to survive in the resource-limited ecosystem of the Dead Sea, Haloarcula marismortui has been reported to have a unique dual-BR system, consisting of HmBRI and HmBRII, instead of only a single BR in a cell (solo-BR). The contribution of this dual-BR system to survival was investigated. First, native H. marismortui and H. salinarum cells were tested in water that had been adjusted to mimic the conditions of Dead Sea water. These archaea were shown to accumulate protons and reduce pH in their periplasmic regions, which disabled further proton transportation functionality in H. salinarum but not in H. marismortui. Then, pH-dependent photocurrent measurements using purified BR proteins demonstrated that HsBR and HmBRI were functional at pH > 5.0 and that HmBRII was functional at pH > 4.0. Our results indicate that the dual-HmBR system is composed of two BRs with different optimal functional pH ranges and together they maintain light-driven proton transport activity under pH > 4.0, which might contribute the survival of H. marismortui under the acidic pH of the Dead Sea.

Cell-Sized Lipid Vesicles as Artificial Antigen-Presenting Cells for Antigen-Specific T Cell Activation.

In this study, efficient T cell activation is demonstrated using cell-sized artificial antigen-presenting cells (aAPCs) with protein-conjugated bilayer lipid membranes that mimic biological cell membranes. The highly uniform aAPCs are generated by a facile method based on standard droplet microfluidic devices. These aAPCs are able to activate the T cells in peripheral blood mononuclear cells, showing a 28-fold increase in interferon gamma (IFNγ) secretion, a 233-fold increase in antigen-specific CD8 T cells expansion, and a 16-fold increase of CD4 T cell expansion. The aAPCs do not require repetitive boosting or additional stimulants and can function at a relatively low aAPC-to-T cell ratio (1:17). The research presents strong evidence that the surface fluidity and size of the aAPCs are critical to the effective formation of immune synapses essential for T cell activation. The findings demonstrate that the microfluidic-generated aAPCs can be instrumental in investigating the physiological conditions and mechanisms for T cell activation. Finally, this method demonstrates the feasibility of customizable aAPCs for a cost-effective off-the-shelf approach to immunotherapy.

Fluorescence-based assay probing regulator of G protein signaling partner proteins.

The regulator of G protein signaling (RGS) proteins are one of the essential modulators for the G protein system. Besides regulating G protein signaling by accelerating the GTPase activity of Gα subunits, RGS proteins are implicated in exerting other functions; they are also known to be involved in several diseases. Moreover, the existence of a single RGS protein in plants and its seven-transmembrane domain found in 2003 triggered efforts to unveil detailed structural and functional information of RGS proteins. We present a method for real-time examination of the protein-protein interactions between RGS and Gα subunits. AtRGS1 from plants and RGS4 from mammals were site-directedly labeled with the fluorescent probe Lucifer yellow on engineered cysteine residues and used to interact with different Gα subunits. The physical interactions can be revealed by monitoring the real-time fluorescence changes (8.6% fluorescence increase in mammals and 27.6% in plants); their correlations to functional exertion were shown with a GTPase accelerating activity assay and further confirmed by measurement of K(d). We validate the effectiveness of this method and suggest its application to the exploration of more RGS signaling partner proteins in physiological and pathological studies.

Overexpression of Different Types of Microbial Rhodopsins with a Highly Expressible Bacteriorhodopsin from Haloarcula marismortui as a Single Protein in E. coli.

Microbial rhodopsins (M-Rho) are found in Archaea, Bacteria and some species of Eukarya and serve as light-driven ion pumps or mediate phototaxis responses in various biological systems. We previously reported an expression system using a highly expressible mutant, D94N-HmBRI (HEBR) from Haloarcula marismortui, as a leading tag to assist in the expression of membrane proteins that were otherwise difficult to express in E. coli. In this study, we show a universal strategy for the expression of two M-Rho proteins, either the same or different types, as one fusion protein with the HEBR system. One extra transmembrane domain was engineered to the C-terminal of HEBR to express another target M-Rho. The average expression yield in this new system reached a minimum of 2 mg/L culture, and the maximum absorbance of the target M-Rho remained unaltered in the fusion forms. The fusion protein showed a combined absorbance spectrum of a lone HEBR and target M-Rho. The function of the target M-Rho was not affected after examination with functional tests, including the photocycle and proton pumping activity of fusion proteins. In addition, an otherwise unstable sensory rhodopsin, HmSRM, showed the same or even improved stability under various temperatures, salt concentrations, and a wide range of pH conditions. This HEBR platform provides the possibility to construct multi-functional, stoichiometric and color-tuning fusion proteins using M-Rho from haloarchaea.

A transducer for microbial sensory rhodopsin that adopts GTG as a start codon is identified in Haloarcula marismortui.

Microbial sensory rhodopsins are known to mediate phototaxis, and all of the known sensory rhodopsins execute this function with a specific cognate transducer that has two-transmembrane (2-TM) regions. In the genome of Haloarcula marismortui, a total of six rhodopsin genes were annotated, and we previously showed three of them to be the ion type and suggested the other three as sensory type, even though the candidate transducer gene, htr, for HmSRI was missing the 2-TM region that is found in all of the other known transducers. Here we showed this htr gene featured a preceding 2-TM region when the alternative start codon GTG located 291 nucleotides upstream of the original annotated open reading frame (ORF) was introduced and it is named as htrI in this study. Overexpression of HmHtrI exhibited it existed as a membrane protein and several biophysical assays confirmed it functionally interacted with HmSRI. Together with our previous reverse-transcriptase-PCR results and phototaxis measurements, the new ORF of original predicted soluble htr gene product was a membrane protein with a 2-TM region, HmHtrI; and it serves as the cognate transducer for HmSRI. HmHtrI therefore is the first transducer for the sensory rhodopsin adopted start codon other than ATG.