Biochemical Analysis of Microbial Rhodopsins

Julia A. Maresca1, Jessica L. Keffer1, Kelsey J. Miller2

1 University of Delaware, Department of Civil and Environmental Engineering, Newark, Delaware, 2 University of Delaware, Department of Biological Sciences, Newark, Delaware
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1F.4
DOI:  10.1002/cpmc.5
Online Posting Date:  May, 2016
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Abstract

Ion‐pumping rhodopsins transfer ions across the microbial cell membrane in a light‐dependent fashion. As the rate of biochemical characterization of microbial rhodopsins begins to catch up to the rate of microbial rhodopsin identification in environmental and genomic sequence data sets, in vitro analysis of their light‐absorbing properties and in vivo analysis of ion pumping will remain critical to characterizing these proteins. As we learn more about the variety of physiological roles performed by microbial rhodopsins in different cell types and environments, observing the localization patterns of the rhodopsins and/or quantifying the number of rhodopsin‐bearing cells in natural environments will become more important. Here, we provide protocols for purification of rhodopsin‐containing membranes, detection of ion pumping, and observation of functional rhodopsins in laboratory and environmental samples using total internal reflection fluorescence microscopy. © 2016 by John Wiley & Sons, Inc.

Keywords: proton pumping; retinal; rhodopsin; spectroscopy; TIRF microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Membrane Purification to Enrich Rhodopsin Fraction
  • Basic Protocol 2: Monitoring Proton Pumping in Rhodopsin‐Containing Cells
  • Basic Protocol 3: TIRF Microscopy of Rhodopsin‐Containing Cells
  • Support Protocol 1: Preparation of Gelatin‐Coated Coverslips
  • Support Protocol 2: Preparation of Unfixed, Unstained Bacterial cells for Tirf Microscopy
  • Support Protocol 3: Preparation of Fixed cells From an Axenic Culture for Tirf Microscopy
  • Support Protocol 4: Preparation of Environmental Water Samples for Tirf
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Membrane Purification to Enrich Rhodopsin Fraction

  Materials
  • Rhodopsin‐expressing bacterial cells of interest
  • Appropriate medium for bacterial cells of interest (see annotation to step 1, below)
  • “Sweet” buffer (see recipe)
  • Lysozyme: 50 mg/ml lysozyme in sterile distilled, deionized H 2O, freshly prepared
  • “Salt” buffer (see recipe)
  • BOG buffer: 3% beta‐octylglucopyranoside (BOG) in 10 mM HEPES, pH 7.1
  • Orbital shaker
  • Centrifuge/microcentrifuge (e.g. Thermo Scientific Sorvall Legend X1R, with rotors 75003655 and 75003652 for 50‐ml tubes and microcentrifuge tubes, respectively)
  • Probe sonicator (e.g. Fisher Scientific sonic dismembrator model FB‐120, with probe model CL‐18)

Basic Protocol 2: Monitoring Proton Pumping in Rhodopsin‐Containing Cells

  Materials
  • Rhodopsin‐expressing bacterial cells of interest
  • Appropriate medium for bacterial cells of interest (see annotation to step 1, below, and annotation to step 1 of protocol 1)
  • Pumping buffer (see recipe), freshly prepared
  • Programmable timer (e.g., Leviton LT112 Digital Plug‐In Timer)
  • 250‐W halogen lamp (e.g., Utilitech 250‐Watt Portable Work Light)
  • 400‐ml beaker
  • Magnetic stir plate and small magnetic stir bars (5 to 8 mm × 1.5 mm)
  • Piece of clear, hard plastic approximately 8 in. × 10 in.
  • “Floater” for microcentrifuge tubes (e.g., Ted Pella floating tube rack, cat no. 20831‐8, or USA Scientific floating foam rack, cat. no. 9138‐7540)
  • Micro pH electrode (for sample sizes <1 ml, e.g., Mettler Toledo inLab Micro pH electrode)
  • Data logger (must be able to connect to pH electrode and store time and pH data, e.g., Sper Scientific pH SD card data logger)

Basic Protocol 3: TIRF Microscopy of Rhodopsin‐Containing Cells

  Materials
  • Slide prepared as described in protocol 4 and sample(s) prepared as described in Support Protocols protocol 52 to protocol 74
  • Immersion oil with a refractive index of 1.518 (e.g., Immersol 518 F; Zeiss, cat. no. 444960)
  • Control samples (see Critical Parameters for protocol 3 for details on suggested control samples)
  • Fluorescence microscopy system capable of TIRF: our system is a lab‐built Zeiss Observer.A1 microscope; for imaging of bacteria, we use a 100/1.46‐numerical‐aperture (NA) oil immersion lens, with an additional × 2 magnification after the tube lens
  • Lasers:
    • 405 nm (Coherent Cube), for imaging DAPI‐stained cells
    • 488 nm (Coherent Sapphire), for imaging carotenoid‐ and rhodopsin‐containing cells
    • 561 nm (Coherent Sapphire), for imaging rhodopsin‐containing cells
    • 641 nm (Coherent Cube), for imaging chlorophyll a‐containing cells
  • Peltier‐cooled (−75°C) charge‐coupled device (CCD) camera: for example, Andor iXON DU897 electron‐multiplying CCD (eMCCD) camera, or Princeton Instruments Excelon ProEM512 CCD camera
  • Emission filter: 446/523/600/677 nm BrightLine quad‐band bandpass filter (Semrock, Inc., cat. no. FF01‐446/523/600/677‐25)
  • Computer equipped with NIH ImageJ

Support Protocol 1: Preparation of Gelatin‐Coated Coverslips

  Materials
  • N hydrochloric acid
  • 95% ethanol
  • Gelatin (Sigma, cat. no. C6144)
  • Chromium (III) sulfate hydrate (Cr 2O 12S 3; Strem Chemicals, cat. no. 9302414)
  • Coverslips (Nunc 155409)
  • Slide chamber (e.g., SPI slide mailer; SPI, cat. no. 01253‐FA)
  • Probe sonicator (e.g. Fisher Scientific sonic dismembrator model FB‐120, with probe model CL‐18)
  • Orbital shaker
  • Slide rack
  • UV light (e.g., in laminar‐flow hood)
  • Forceps

Support Protocol 2: Preparation of Unfixed, Unstained Bacterial cells for Tirf Microscopy

  Materials
  • Rhodopsin‐expressing bacterial cells of interest
  • Bacterial growth medium (see Basic Protocols protocol 11 and protocol 22)
  • Refrigerated centrifuge
  • Forceps
  • Cleaned, gelatin‐coated coverslips ( protocol 4)
  • Clean glass microscope slides
  • Nail polish or other sealant

Support Protocol 3: Preparation of Fixed cells From an Axenic Culture for Tirf Microscopy

  Materials
  • Rhodopsin‐expressing bacterial cells of interest
  • Bacterial growth medium (see Basic Protocols protocol 11 and protocol 22)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 20% (w/v) paraformaldehyde in PBS
  • NucBlue Fixed Cell ReadyProbes Reagent (Life Technologies, cat. no. R37606)
  • Nail polish or other sealant
  • Centrifuge
  • Forceps
  • 25‐mm, 0.2‐μm polycarbonate filters (EMD Millipore)
  • Filter holder with stainless steel support for 25‐mm filters (e.g., EMD Millipore, cat. no. XX1002540)
  • Vacuum pump
  • Cleaned, gelatin‐coated coverslips ( protocol 4)
  • Clean glass microscope slides

Support Protocol 4: Preparation of Environmental Water Samples for Tirf

  Materials
  • Environmental water sample of interest
  • 20% (w/v) paraformaldehyde in phosphate‐buffered saline (PBS; see appendix 2A)
  • NucBlue Fixed Cell ReadyProbes Reagent (Life Technologies, catalog R37606)
  • Nail polish or other sealant
  • Larger‐pore‐size filter (3‐ to 5‐μm pore size; optional)
  • Forceps
  • 25‐mm, 0.2‐μm polycarbonate filters (EMD Millipore)
  • Filter holder with stainless steel support for 25‐mm filters (e.g., EMD Millipore, cat. no. XX1002540)
  • Vacuum pump
  • Cleaned, gelatin‐coated coverslips ( protocol 4)
  • Clean glass microscope slides
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Figures

Videos

Literature Cited

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