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

Summary

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.

Abstract

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.

Introduction

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 (<....

Protocol

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 medium (2 x 500 mL batches) and grow for 2 - 3 hours to OD600 = 0.6 - 0.8 (37 °C, 200 rpm ).
4. Cool cultures on ice. Induce with 250 µM....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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