E. coli Expression and Purification of Microbial and Viral Rhodopsins.

Microbial rhodopsins have become an indispensable tool for neurobiology. Of thousands of identified microbial rhodopsins, a minute fraction has been studied so far and they have shown remarkable functional diversity suggesting more great promises that this large family holds. Effective production of recombinant microbial and viral rhodopsins is a prerequisite for the success of functional and structural studies of these proteins. Escherichia coli (E. coli) are suitable for high yield expression of many of microbial and viral rhodopsins and they facilitate rapid exploration of this large protein family.

True-atomic-resolution insights into the structure and functional role of linear chains and low-barrier hydrogen bonds in proteins.

Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited. The challenge is that their 'visualization' requires ultrahigh-resolution structures of the ground and functionally important intermediate states to identify proton translocation events and perform their structural assignment. Our true-atomic-resolution structures of the light-driven proton pump bacteriorhodopsin, a model in studies of proton transport, show that CHBs and LBHBs not only serve as proton pathways, but also are indispensable for long-range communications, signaling and proton storage in proteins. The complete picture of CHBs and LBHBs discloses their multifunctional roles in providing protein functions and presents a consistent picture of proton transport and storage resolving long-standing debates and controversies.

Viral rhodopsins 1 are an unique family of light-gated cation channels.

Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na+/K+-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca2+ ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca2+ permeability.

Unique structure and function of viral rhodopsins.

Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists.

Structure and mechanisms of sodium-pumping KR2 rhodopsin.

Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.

UAVs, Hyperspectral Remote Sensing, and Machine Learning Revolutionizing Reef Monitoring.

Recent advances in unmanned aerial system (UAS) sensed imagery, sensor quality/size, and geospatial image processing can enable UASs to rapidly and continually monitor coral reefs, to determine the type of coral and signs of coral bleaching. This paper describes an unmanned aerial vehicle (UAV) remote sensing methodology to increase the efficiency and accuracy of existing surveillance practices. The methodology uses a UAV integrated with advanced digital hyperspectral, ultra HD colour (RGB) sensors, and machine learning algorithms. This paper describes the combination of airborne RGB and hyperspectral imagery with in-water survey data of several types in-water survey of coral under diverse levels of bleaching. The paper also describes the technology used, the sensors, the UAS, the flight operations, the processing workflow of the datasets, the methods for combining multiple airborne and in-water datasets, and finally presents relevant results of material classification. The development of the methodology for the collection and analysis of airborne hyperspectral and RGB imagery would provide coral reef researchers, other scientists, and UAV practitioners with reliable data collection protocols and faster processing techniques to achieve remote sensing objectives.

A Novel Methodology for Improving Plant Pest Surveillance in Vineyards and Crops Using UAV-Based Hyperspectral and Spatial Data.

Recent advances in remote sensed imagery and geospatial image processing using unmanned aerial vehicles (UAVs) have enabled the rapid and ongoing development of monitoring tools for crop management and the detection/surveillance of insect pests. This paper describes a (UAV) remote sensing-based methodology to increase the efficiency of existing surveillance practices (human inspectors and insect traps) for detecting pest infestations (e.g., grape phylloxera in vineyards). The methodology uses a UAV integrated with advanced digital hyperspectral, multispectral, and RGB sensors. We implemented the methodology for the development of a predictive model for phylloxera detection. In this method, we explore the combination of airborne RGB, multispectral, and hyperspectral imagery with ground-based data at two separate time periods and under different levels of phylloxera infestation. We describe the technology used-the sensors, the UAV, and the flight operations-the processing workflow of the datasets from each imagery type, and the methods for combining multiple airborne with ground-based datasets. Finally, we present relevant results of correlation between the different processed datasets. The objective of this research is to develop a novel methodology for collecting, processing, analising and integrating multispectral, hyperspectral, ground and spatial data to remote sense different variables in different applications, such as, in this case, plant pest surveillance. The development of such methodology would provide researchers, agronomists, and UAV practitioners reliable data collection protocols and methods to achieve faster processing techniques and integrate multiple sources of data in diverse remote sensing applications.

An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin.

Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.