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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Published: July 17th, 2018

DOI:

10.3791/58157

1Cellular Imaging Group, Wellcome Centre Human Genetics, University of Oxford, 2Division of Structural Biology, Wellcome Centre Human Genetics, University of Oxford, 3Dynamic Structural Virology Group, Biocruces Health Research Centre, 4IKERBASQUE, Basque Foundation for Science

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Protein-protein Interactions In Vitro

Traditionally, crystallography and nuclear magnetic resonance experiments combined with cryo-electron microscopy (cryoEM) are the technologies chosen to accurately describe the three-dimensional architecture of proteins and to infer their function by scrutinizing their high resolution structural details. Proteins, however, are not static structures and can undergo a variety of conformational changes and vibrations in time and space. This is why structural information from crystallographic or CryoEM data needs to be complemented with other techniques (<....

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1. FKBP12 F36V -mVenus Purification

  1. Transform (DE3) pLysS cells with pET22b vector containing monomerized human FKBP12F36V12 and N-terminal His6 and mVenus tags (vector available on request). Plate cells onto LB agar supplemented with 50 µg/mL Ampicillin and 34 µg/mL Chloramphenicol.
  2. Transfer transformed colonies into 100 mL LB starter culture and grow for 16 - 20 hours at 37 °C with shaking.
  3. Dilute dense starter culture (OD600 >1) 1:100 in LB med.......

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Detrending and monomeric brightness

Once the data has been acquired, one can start the brightness calculations to determine the oligomeric state of the protein of interest in the solution. Even if the effect of bleaching in solution may not be as drastic as it can be in vivo, the intensity trace will still probably not have stationary mean, possibly due to photophysical effects related to the fluorophor.......

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N&B is a technique to detect multimerization using commercial light scanning confocal microscopes equipped with digital detectors. This approach is quite attractive compared to single point FCS, FCCS, and smFRET because it is calibration free and the brightness calculation is straightforward and concentration independent6. It is of major importance, however, to correct for bleaching and long-term intensity fluctuations before performing brightness calculations9; a sligh.......

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This work has been supported by Wellcome Trust grant 105278/Z/14/2 to R.N. The Wellcome Trust Centre for Human Genetics is funded by Wellcome Trust Core Award 203852/Z/16/2. Work in the C.S. group is supported by Cancer Research UK (C20724/A14414), and the European Research Council under the European Union's Horizon 2020 Research and Innovation Programme Grant 647278.

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Name Company Catalog Number Comments
RosettaTM (DE3) pLysS cells Novagen 70956-3
Ampicillin Sigma Aldrich PubChem Substance ID 329824407
Chloramphenicol Sigma Aldrich PubChem Substance ID: 24892250
LB starter culture QIAGEN
LB medium QIAGEN https://www.sigmaaldrich.com/content/dam/sigma-aldrich/head/search/external-link-icon.gif
IPTG Sigma Aldrich PubChem Substance ID 329815691
IMAC buffer Medicago 09-1010-10
EDTA-free protease inhibitors  Sigma Aldrich 11873580001
TALON resin Clonetech
Nickel sepharose GE Healthcare
S200 16/60 column GE Healthcare
Glass bottom 8 well observation dish Ibidi 80827

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