Materials and Reagents

Tips: Sterile pipette tips (low-retention tips)
Falcon, 15-ml
Sterile microcentrifuge tubes (1.5-ml)
Wash bottle
Liquid discard beaker
Kimwipe tissue paper (Kimberly-Clark, catalog number: 34120)
Microcentrifuge tube rack
Falcon rack
Fluorescence cuvette (3.0 mm path length) (Hellma Analytics, catalog number: Z802336)
0.22-μm filter (Millex, catalog number: SLGP099RS)
10-ml syringe
MilliQ water (deionized water)
Methanol (100%) (HIMEDIA, catalog number: AS061-2.5L)
Guanidine hydrochloride (MP, catalog number: 105696)
Magnesium chloride (HIMEDIA MB, catalog number: 040-500G)
Potassium chloride (Sigma, catalog number: 793590)
Tris (ultra-pure) (MP, catalog number: 103133)
HCl (HMEDIA, catalog number: AS004-2.5L)
ATP (Sigma, catalog number: A2383)
DTT (MP, catalog number: 100597)
Purified protein (sGFP)
Purified chaperones and co-chaperones (GroEL, GroES)
sGFP (Sadat et al., 2020)
Buffer A (see Recipes)

Equipment

Pipettes: 2.5-μl, 10-μl, 200-μl, 1-ml
Centrifuge
Fluorescence spectrophotometer (Fluorolog 3 Spectrofluorometer, Jobin Yvon, Horiba)
Dry bath
pH meter

Procedure

sGFP spontaneous refolding
1. Protein Unfolding
  Add 2.5 μl sGFP (20 μM) to a microcentrifuge tube containing 7.5 μl 8 M GuHCl (final GuHCl concentration in the unfolding reaction is 6 M) in buffer A (see Recipes). Place the tube in a dry bath for 1 h at 25°C. This will lead to complete unfolding of the protein.
2. Protein Refolding
  1. Switch on the Fluorolog instrument (Fluorescence spectrofluorometer). Keep the instrument switched ON for 45 min prior to fluorescence measurement, so that the lamp is uniformly heated and fluctuations are minimal.
  2. Set the instrument to emission spectra mode. Keep the excitation wavelength at 350 nm and the emission spectra in the 365-430 nm range. After 45 min, add MilliQ H₂O to a quartz microcuvette and take a Raman Spectra. If you get an emission peak at 397 nm, the spectrofluorometer is working fine.
  3. Set the instrument to kinetic acquisition mode. Set the excitation wavelength to 480 nm and the emission wavelength to 515 nm. Set to anti-photobleaching mode. Set the collection interval to 20 s and the integration time to 0.7 s. The total time for the kinetics is set to 1,800 s. Set the temperature to 25°C. The slit-width is kept at 2 nm for excitation and 5 nm for emission.
  4. Clean a quartz cuvette (Hellma Analytics, 10 mm path length) with 100% methanol and then MilliQ water, and wipe using a kimwipe. Take 2 μl (200 nM final) completely unfolded protein, dilute 100-fold in Buffer A (198 μl), and mix thoroughly by pipetting (total reaction volume 200 μl) in the cuvette.
  5. Start the kinetics acquisition as soon as the mixing is finished (keep an equal delay in this step).
  6. sGFP refolding typically follows a two-state pathway. The unfolded proteins are refolded in refolding buffer to their native state in a single step. In the refolding experiment, we monitor the increase in GFP fluorescence by exciting the refolding reaction at 480 nm and measuring the fluorescence emission at 515 nm. GFP fluoresces only after folding into its native state; therefore, GFP fluorescence is the readout for the amount of folded protein in our refolding experiment. The refolding kinetics trace obtained after the refolding reaction can be best fitted using nonlinear regression by a single exponential kinetics equation. The kinetics data are analyzed using the Origin software.
A mathematical model to fit the refolding kinetics:
The model for fitting the data obtained from refolding experiments is a mathematical equivalent of two-state folding kinetics. The one-phase exponential decay equation is as follows:

where y₀ is the offset, A₁ is the amplitude, and t₁ is the time constant. The apparent rate of the refolding reaction is obtained by taking the reciprocal of the time constant. A typical refolding kinetics plot is shown in Figure 1a.

Chaperonin-assisted sGFP refolding
1. Protein Unfolding
  Unfold sGFP as described earlier in Step A1.
2. Protein Refolding
  a, b, c follow steps A2a, b, c as described for spontaneous refolding.
  1. Take 2 μl (200 nM) completely unfolded protein and dilute 100-fold in Buffer A containing GroEL (400 nM tetradecamers of GroEL). Add GroES (800 nM heptamers of GroES), such that the ratio of substrate to GroEL toGroES is 1:2:4. The refolding is initiated by the addition of 2 mM ATP (final concentration); the total reaction volume is 200 μl. Mix the reaction thoroughly by pipetting slowly.
  2. Start the kinetics acquisition as soon as the mixing is finished (keep an equal delay in this step).
    A representative refolding kinetics plot is shown in Figure 1a. Figure 1b, 1c, 1d explains the various steps involved in the data analysis for calculating the refolding rate. 1/t1 gives the refolding rate/second.
  3. Same as Step A2f.
Figure 1. sGFP refolding plot. a.Plot showing the kinetics of sGFP refolding as measured by an increase in GFP fluorescence. sGFP was unfolded in 6 M GuHCl in Buffer A at 25°C for 1 h. Subsequently, unfolded sGFP was refolded by 100-fold dilution in Buffer A. b. The output screen after refolding. c. This screen appears by clicking on analysis after selecting the columns for time and fluorescence. d. This screen appears after the fitting by selecting ‘expdec1’ in the function section.

Notes

While this method can be used for monitoring the refolding of any fluorescent protein, we must keep in mind that only those fluorescent proteins that start to fluoresce as soon as they are folded are exclusively suitable for measuring refolding kinetics using the described protocol. As described, sGFP fluoresces as soon as it is folded into its native state; hence, we actually measure the rate of increase in sGFP fluorescence, which corresponds to the refolding rate of the protein. Therefore, this is an indirect method of calculating the refolding rate of sGFP. We assume that there are no such limitations in using this method for measuring the refolding rate of other fluorescent model proteins by similar spontaneous or chaperonin-assisted in vitro refolding assays. This protocol can be easily modified, especially the instrument settings (slit-width, excitation-emission wavelengths, total time of kinetic measurements), time of unfolding, concentrations of GuHCl, and other parameters depending on the properties of the model fluorescent protein used. In the case of chaperone-assisted refolding experiments, the molar ratio of substrate to chaperones in the refolding buffer must be optimized for the individual chaperone/co-chaperones used for the refolding assay.

Recipes

Buffer A
20 mM Tris pH 7.4
20 mM KCl
5 mM MgCl₂
2 mM DTT
Filtered using a 0.22-μm filter and a 10-ml syringe

Acknowledgments

K.M. acknowledges the funding from the Department of Biotechnology (DBT), Government of India (grant number BT/PR28386/BRB/10/1671/2018) and the Science and Engineering Research Board (SERB), Government of India, for Core Research Grant (SERB/CRG/2019/006281) and SNU core funding. The work in the IGIB Lab was supported by a Swarnajayanthi Fellowship Grant from DST, and partly by BSC0124 from CSIR. Instrument support was also obtained from Wellcome Trust-DBT India Alliance and CSIR. KM, acknowledges SNU, and AS and ST acknowledge CSIR and CSIR-IGIB for infrastructural support. AS acknowledges DBT.

Competing interests

The authors declare no competing interests.

References

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