This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.
Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, ΔG°H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.
1. Preparation of ~50 nm Diameter Small Unilamellar Vesicles (SUVs) for Membrane Protein Folding
2. Sample Preparation for Initial Fluorescence Unfolding Curve
3. Measurement of Fluorescence Spectra
Tryptophan fluorescence of each sample and blank is measured using a steady-state fluorometer. Fluorescence spectra should be recorded with excitation wavelength of 290 nm to avoid excitation of tyrosine residues, and scanned from 305 to 500 nm. Typical entrance and exit bandpass is 3 nm. The wavelength increment and integration time can be optimized for signal-to-noise ratio. Typical values for wavelength increment and integration time are 1 nm/step and 0.5 sec/step, respectively. Membrane proteins generally fold into synthetic lipids only when the temperature is above the bilayer phase transition temperature. Therefore, in this case of DMPC, the temperature of the sample is held constant at 30 °C. A microvolume fused silica cuvette with 160 μL capacity is utilized in these experiments.
4. Generation of Initial Unfolding Curve and Estimation of Gibbs Free Energy of Membrane Protein Unfolding
5. Optimized Unfolding Curve for More Precise Measurement of Gibbs Free Energy of Unfolding.
6. Representative Results:
Sample | final [Urea], M | stock protein (μL) | stock lipid (μL) | stock 10 M urea (μL) | stock buffer (μL) |
1 | ~ 0.16 | 4 | 40 | 0 | 156 |
2 | 1 | 4 | 40 | 20 | 136 |
3 | 2 | 4 | 40 | 40 | 116 |
4 | 3 | 4 | 40 | 60 | 96 |
5 | 4 | 4 | 40 | 80 | 76 |
6 | 5 | 4 | 40 | 100 | 56 |
7 | 6 | 4 | 40 | 120 | 36 |
8 | 7 | 4 | 40 | 140 | 16 |
Table 1. Volume of stock solutions required to make fluorescence samples
Figure 1. Tryptophan fluorescence spectra of ~ 5 μM representative membrane protein that contains a single tryptophan residue. (A) Raw fluorescence spectra of protein (spectrum A from 4.2); (B) Raw fluorescence spectra of blank (spectrum B from 4.2); (C) Corrected spectrum from 4.2.
Figure 2. Corrected tryptophan fluorescence spectra of membrane protein from Figure 1 for numerous urea concentrations.
Figure 3. Unfolding curve obtained from data in Figure 2, with fit to equation in 4.4. The Gibbs free energy is calculated according to section 4.5.
The current protocol describes the generation of unfolding curves of membrane-associated proteins and peptides that contain tryptophan residues. Here, it is assumed that the tryptophan fluorescence reflects whether the protein is folded and inserted into synthetic lipid vesicles, or unfolded in solution. Additional assumptions, such as two-state folding and linear dependence of free energy with denaturant concentration, are made in the current report; modification of these assumptions result in different equations.2 The experimental protocol can easily be modified to utilize different spectroscopic observables, such as circular dichroism, absorption, or fluorescence from an extrinsic dye,3 as well as gel electrophoresis.4 Additionally, different denaturants, such as guanidinium, acidic solutions, or temperature, as well as alternative lipids/vesicles, such as anionic lipids, large unilamellar vesicles, or micelles, can be utilized.5,6 Finally, the protocol can be applied to any membrane protein, membrane-associated peptide, and soluble protein/peptide that meets the criteria of reversible folding, and that exhibits a spectroscopic or other observable marker for different conformational states. In the case of soluble proteins, vesicles are obviously omitted from the protocol.
Gibbs free energy of unfolding provides insight into the noncovalent interactions that stabilize biomolecules. In our lab, we have measured unfolding curves for mutants of Outer Membrane Protein A (OmpA) that contain single tryptophan residues in different locations, and reported that the mutants were destabilized relative to the wild-type protein that contains five native tryptophan residues.7 These unfolding studies are complemented by other techniques, including vibrational spectroscopy, to elucidate molecular details of, for example, hydrogen bonding and pairwise interactions that underlie the variation in stability. In summary, the relatively straightforward technique of unfolding curves can be applied to membrane and soluble proteins to reveal thermodynamics associated with protein folding.
The authors have nothing to disclose.
We thank Beijing Wu for use of her data. This work was supported by an NSF CAREER award to J.E.K.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
DMPC | Avanti Polar Lipids | 850345C | ||
Urea | MP Biochemicals | 04821527 | ||
Potassium Phosphate Dibasic | Fisher | P288 | ||
Potassium Phosphate Monobasic | Fisher | P285 |