Application Note

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Measuring the conformational stability of membrane proteins using the UNit

Geoffrey Platt1, Vincent Postis2,3, Zhenyu Hao2, Tony Palmer2 and Steve Baldwin2 1Unchained Labs; 2Faculty of Biological Sciences, University of Leeds; 3Faculty of Health and Social Sciences, Leeds

Beckett University

Introduction Use of extrinsic dyes to study protein unfolding Measurement of thermal stability is a valuable tool for

assessing the suitability of proteins to a wide range of

applications, including their use as therapeutic drugs or

food additives. Indeed, researchers commonly require

practical methods to rapidly screen conditions that

afford the best environment for a particular protein. The

UNit provides a route to obtaining such thermal stability

information in a high throughput manner by probing

intrinsic fluorescence and static light scattering changes

of up to 48 samples simultaneously. In certain instances,

it is helpful to expand the repertoire of optical probes

used to assess the stability of proteins, such as by adding

fluorescent dyes (e.g. 1-Anilinonaphthalene-8-Sulfonic

Acid (ANS), SYPRO orange) that are sensitive to the

amount of exposed hydrophobic residues. In other cases,

the user can monitor the status of additional sensors

such as the fluorophore present in green fluorescent

protein (GFP). The UNit with an optional 375 nm laser

has been designed to measure the thermal stability of

both globular and integral membrane proteins using an

extrinsic dye that covalently and specifically interacts

with the unfolded form of the proteins. This method

should be of particular importance for gaining stability

information for membrane proteins.

Membrane protein stability Approximately 30 % of the human genome encodes

membrane proteins, which include receptors, transport

proteins and enzymes. In their native state, they are

embedded in, or attached to, the complex environment

of a lipid bilayer of a cell or organelle membrane. The

majority of known pharmaceutical targets are membrane

proteins, hence the success of drug-design efforts will

depend on their structural characterization. It is not

often possible, however, to use typical biophysical

techniques such as X-ray crystallography or NMR to

study these proteins in their native environment.

Therefore, extraction of the proteins from their

membranes with detergents is required before structural

analyses can be performed. This process often leads to

destabilization, aggregation or unfolding [1] and therefore

establishing stability conditions of a protein in a

detergent-solubilized state makes a key contribution to

the successful crystallization of membrane proteins.

A number of techniques, including light scattering [2] and

addition of extrinsic fluorescent dyes that are reactive to

exposed hydrophobic regions,[3] have been utilized to

identify the optimum conditions for maintaining the

stability of membrane proteins. Unfortunately, there are

limitations to the use of extrinsic fluorescent dyes as

they partition in the presence of free detergent micelles

and protein-detergent complexes resulting in high

background fluorescence. Furthermore, in one study that

measured the extrinsic fluorescence change for such

dyes in the presence of four membrane proteins it was

Application Note

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Figure 1A: Structure of 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). 1B: Structure of monomeric -

lactoglobulin (-LG, PDB: 3NPO)[9] – sidechains of Trp19 and Trp61 are displayed in violet; Cys 121 is displayed in red and disulphide

bonds are shown in yellow. 1C: Structure of Vibrio cholera concentrative nucleoside transporter (VcCNT, PDB: 3TIJ)[13] – Trp

sidechains are displayed in violet, Cys sidechains are displayed in red and the position of the membrane bilayer is indicated with grey

boxes.

observed that only those proteins with large surface-

hydrophilic extra-membranous regions showed clear

transitions.[3]

To overcome these limitations, a novel method has been developed using a highly reactive thiol-specific fluorescent dye called 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM, Figure 1A).[4] This fluorochrome greatly increases its fluorescenceintensity upon covalent reaction with cysteine (Cys) residues [5] and hence acts as a probe for the exposure of these residues and for protein unfolding in general. This method is particularly useful for membrane proteins as cysteines have been noted to be frequently located at helix-helix interaction sites and should therefore act as sensors for their overall structural integrity.[6]

A B

C

To demonstrate the applicability of the CPM method for

measuring thermal unfolding of proteins, different

classes of proteins have been studied using the UNit

with optional 375 laser. Firstly, a globular protein,

bovine -lactoglobulin (-LG, Figure 1B), and secondly,

two related integral membrane proteins that are part of

the concentrative nucleoside transporter family from E.

coli (NupC) and V. cholera (VcCNT, Figure 1C) were

examined.

Both the conformational thermal stability of these

proteins and their propensity to aggregate was studied

using the UNit with optional 375 laser to measure

intrinsic fluorescence, CPM-derived fluorescence and

static light scattering. The methods used allowed the

demonstration of a number of the features of the UNit

with optional 375 laser including:

Application Note

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Use of three lasers

Multiple device settings for each well

Different device settings for individual wells

Materials and Methods Materials The membrane proteins (NupC and VcCNT) were expressed and purified at the University of Leeds and were kindly supplied by Prof. Steve Baldwin. Lyophilized β lactoglobulin (β-LG, cat. no. L3908) waspurchased from Sigma-Aldrich (Poole, UK). 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM, cat. no. D346) and dimethylsulfoxide (DMSO) were purchased from Life Technologies, Paisley, UK. All other reagents were purchased from Sigma-Aldrich (Poole, UK).

Sample preparation

-lactoglobulin was studied at 0.2 mg mL-1 (10.8 M) in

50 mM HEPES (pH 7.5), 100 mM NaCl, 0.025 % (w/v) n-

dodecyl-β-D-maltopyranoside (DDM), 5 % (v/v) DMSO in

the presence or absence of 162 M CPM. The

membrane proteins were studied at 0.5 mg mL-1 (~11.5

M NupC, ~11.1 M VcCNT) in 50 mM HEPES (pH 7.4),

150 mM NaCl, 0.05 % (w/v) DDM, 5 % (v/v) glycerol, 0.9

% (v/v) DMSO in the presence or absence of 162 M

CPM. Half of the membrane protein samples included

uridine (added at a final concentration of 1 mM).

time of 600 msec and then separately by the 375 nm

laser (filter 2) for an exposure time of 200 msec.

Data analysis was achieved using the UNit Analysis 2.1

software. The changes in intrinsic fluorescence (excited

using the 266 nm laser) with temperature were

monitored via analysis of the ratio of intensities at 350

and 330 nm. The changes in static light scattering (SLS)

were probed by measuring the intensities of the peaks

at 266, 375 and 473 nm, where appropriate. The

changes in CPM fluorescence (excited using the 375 nm

laser) were monitored by measuring the intensity of the

emission peak centered at 460 nm.

Results Thermal ramps using independent filter settings in

The Unit with additional 375 laser

The UNit with optional 375 laser is designed to

simultaneously measure the fluorescence and SLS of up

to 48 low-volume samples during a temperature ramp.

The instrument houses three lasers, each of which can

be optimized separately by the use of independent

filter settings. In the experiments described here, lasers

at 266, 375 and 473 nm have been used in various

formats to illuminate the samples.

The laser at 266 nm can be used to excite the intrinsic

fluorescence of proteins via the aromatic residues

present, particularly tryptophan (Trp). These residues

are typically buried in the hydrophobic core of native

globular proteins (for example see -LG structure in

Figure 1B) and show a characteristic shift in their

maximum peak emission to longer wavelengths during

unfolding (Figure 2). This conformational unfolding can

be followed easily by plotting the ratio of the emission intensity at 350 nm with 330 nm.

The extrinsic dye CPM has a maximum excitation at 387 nm and can be excited effectively with the 375 nm laserpresent in the UNit with optional 375 laser. The fluorescence of this molecule is very low in solution, butgreatly increases as it covalently reacts with Cys

UNit experiments The data were acquired using the UNit with additional 375 laser running the UNit Client 2.1 software. Each protein sample was loaded in triplicate in the presence and absence of CPM. For β-LG a linear thermal ramp from 25 to 95 °C at a rate of 0.5 °C min-1 was performed, whereas the ramp for the membrane proteins increased from 15 to 95 °C at a rate of 0.3 °C min-1. Multiple device settings were used on each well – the samples were illuminated firstly with the 266 nm and 473 nm lasers (filter 4 and filter 2, respectively) for an exposure

Application Note

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sidechain thiols that become exposed as protein

unfolds to give an emission peak centered at 463 nm

(Figure 2). This reaction is pH-sensitive and has an

optimum rate at pH 6.5-7.5.

Figure 2: Intrinsic fluorescence spectra (excited at 266 nm) of

0.2 mg mL-1 -LG alone at pH 7.5 overlaid with fluorescence

spectra of 0.05 mg mL-1 -LG in the presence of 25 M CPM

(excited at 375 nm).

Separate illumination of the samples with 266 nm and

then 375 nm lasers prevents complications in the

fluorescence data. The intrinsic protein fluorescence

emission peak occurs at a wavelength absorbed by CPM

molecules.

In addition to the fluorescence data, the scattered light

intensity from each of the lasers (266, 375 and 473 nm)

can be used to assess the aggregation state of the

samples.

Effect of CPM on -lactoglobulin thermal unfolding

A linear thermal ramp experiment was performed using

samples of 0.2 mg mL-1 (10.8 M) -LG in the presence

and absence of 162 µM CPM as described in the Materials and Methods. This is the major whey protein of bovine milk and is a member of the lipocalin family, which typically bind and transport small hydrophobic ligands.[7] It is dimeric at neutral pH and is 162 residues in length.[8] Structurally, β-LG is composed of nine anti-parallel β-strands and one α-helix, and contains two

disulphide bonds (Cys66-Cys160; Cys109-Cys119) and

one free cysteine (Cys121).[9] This free cysteine is buried

in the core of the native state and should only be

solvent exposed (and hence CPM-reactive) if the protein

visits an unfolded species.[4] As described earlier the

intrinsic fluorescence of two buried Trp residues also

provides a probe for changes in tertiary structure and

can thus act as a comparative probe for conformational

stability of the protein, allowing the effectiveness of

CPM to be verified.

Each well was illuminated consecutively by light

controlled with different filter settings before moving on

to the next well. The first device setting allowed laser

light at 266 and 473 nm to illuminate the sample (and

provide information on intrinsic fluorescence); the

second device setting illuminated the sample only with

light at 375 nm (to excite the CPM fluorescence). The

data for -LG incubated in the presence of 162 M CPM

are shown in Figure 3 and Table 1.

The intrinsic fluorescence ratio (350 nm:330 nm)

displays a clear transition for the -LG and provides a Tm

value (temperature at which 50 % of the protein is

unfolded) of 64.9 °C. The conformational stability data

obtained for the same samples using CPM also displays

a clear transition with a Tm value of 62.0 °C. The

differences in these values likely reflect the fact that

each method probes a separate part of the protein

molecule as it undergoes the same conformational

change. Indeed it is now known that non-native partially

folded intermediates accumulate during the refolding of

β-LG, meaning that Cys121 and Trp residues may experience changes to their local environment at different stages of the thermal ramp.[10] The similarity of the traces obtained for intrinsic and extrinsic fluorescence confirms that CPM can successfully report on conformational changes in proteins containing buried reduced Cys residues. In the absence of CPM, a transition in intrinsic fluorescence is observed for these data, however, there are no observable changes in the region of the spectrum corresponding to CPM emission as expected (data not shown). The value of Tm

Application Note

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measured in the absence of the dye (Tm 77.5±0.7 °

C) is higher than that in the presence of CPM,

which may indicate that (at least under these

conditions) interaction with the dye reduces the

stability of the protein. Indeed, it has previously

been observed that covalent modification of the

free thiol at Cys121 destabilizes -LG.[11,12] At near

to neutral pH, conjugation of a small molecule,

mercaptopropionic acid, resulted in the Tm value

for -LG being reduced by more than 15 °C.[11]

Similar effects were seen for modification of

Cys121 with 5-thio-2-nitrobenzoic acid, where the

reaction at neutral pH results in formation of a

monomeric molten globule state.[12] This is

attributed to that fact that the presence of larger

adducts on Cys121 lead to disruption of the dimer

interface.

The stability effects of CPM observed for -LG are likely

to be specific to this protein due to the position of the

free Cys near to the dimer interface. The intrinsic

fluorescence measurements in the presence of the dye

confirm that CPM provides an accurate probe of

structural change in this protein. Screening of multiple

formulation conditions in the presence of CPM should

thus provide an informative way of ranking -LG

conformational stabilities.

Two membrane proteins from the concentrative

nucleoside transporter family were measured using the

UNit with optional 375 laser in this study. NupC is a

protein of 43.5 kDa found in E. coli and VcCNT is a

homologous protein of 44.9 kDa from V. cholera.[13]

Both proteins contain multiple tryptophans but NupC

has a single buried Cys residue and VcCNT contains two

Cys residues that remain in the reduced form in the

typical stability measurements based on optical

techniques, especially those using extrinsic dyes that

non-specifically bind to exposed hydrophobic patches

of proteins. Furthermore, the proteins have been

extracted from their natural environment within a lipid

bilayer and are stabilized in the nonionic surfactant

DDM. Use of surfactants can lead to partitioning of the

typical extrinsic dyes used to measure thermal stability

of globular proteins.

Figure 3: Thermal ramp data collected for -LG in the presence

of 162 M CPM. Intrinsic fluorescence data (excited at 266 nm; top) and CPM fluorescence emission data (excited at 375 nm; bottom) were collected for each sample.

Sample Tm (intrinsic

fluorescence, °C) Tm (CPM

fluorescence, °C)

0.2 mg mL-1

LG pH 7.5 64.9±1.7 62.0±0.5

Table 1: Parameters extracted from the experimental data presented in Figure 3.

Application Note

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Measurement of thermal stability of membrane proteins During the thermal ramp of these proteins changes in

conformational state were monitored using both Trp

fluorescence and exposure of the thiol sidechain of Cys

residues to CPM. Aggregation could be monitored using

SLS from the various wavelengths used to illuminate the

samples.

In the past it has been seen that addition of ligands has

increased the success rate of protein purification and

crystallization.[14] This is the case as ligands that interact

preferentially (specifically or non-specifically) with the

native state of a protein increase the thermal

stability.[15] Here, the proteins from the concentrative

nucleoside transporter family were monitored in both

the absence and presence of one of their natural

ligands, the nucleoside uridine.[16]

Figure 4: Thermal ramp data obtained with 0.5 mg mL-1 NupC

in the presence and absence of 1 mM uridine. Measurements

of change in intrinsic fluorescence emission (top) and CPM

fluorescence emission intensity (bottom).

The data obtained for NupC are displayed in Figure 4.

The intrinsic fluorescence (as monitored using the ratio

of intensities at 350 and 330 nm) for this protein does

not indicate a clear unfolding transition for either the

samples in the presence or absence of uridine. This is

also true for the intrinsic fluorescence data obtained for

VcCNT (Figure 5) and can be explained by the fact that

the Trp residues for this protein (and likely the

homologous NupC) are located in surface exposed

positions (Figure 1C). They are found in parts of the

protein that are within the lipid bilayer and hence are in

a hydrophobic environment even though they are

surface exposed. This means that in the purified protein

it is likely that the detergent used (DDM) will screen

these residues in both the unfolded and folded states

meaning that little change will be observed upon

unfolding.

Figure 5: Thermal ramp data obtained with 0.5 mg mL-1

VcCNT in the presence and absence of 1 mM uridine.

Measurements of change in intrinsic fluorescence emission

(top) and CPM fluorescence emission intensity (bottom).

Nup C

Nup C + uridine

VcCNT

VcCNT + uridine

Application Note

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The data acquired for the same samples incubated in

the presence of CPM are displayed in Figure 4 (for

NupC) and Figure 5 (for VcCNT). Unlike the data

acquired using intrinsic fluorescence alone the traces

show a transition for both proteins, indicative of

conformational change monitored by Cys exposure. This

probe allows thermal stability measurements to be

made resulting in Tm values for both proteins (Table 2).

Addition of uridine produces a small increase in the

thermal stability of the proteins, indicating there is an

interaction and suggesting presence of this ligand could

aid purification and crystallization efforts for these

proteins. Indeed, increasing the concentration of

uridine to levels in excess of the KD value (2.6 mM)[17]

would be expected to have a greater impact on stability

of the complexed protein.[15]

Interestingly, the VcCNT samples consistently display

higher CPM emission intensities than do the NupC

samples. This is likely a reflection of the fact that VcCNT

contains twice as many Cys residues as NupC.

In addition to gaining both intrinsic and extrinsic

fluorescence information, the UNit with optional 375

laser also permits the simultaneous collection of SLS

data. The intensity of SLS is monitored with

temperature and reports on the aggregation state of

the samples. As three lasers were used to illuminate the

membrane proteins studied here, it is possible to gain

separate intensities from the 266, 375 and 473 nm

lasers. As the efficiency of the light scattering process

increases significantly and non-linearly as the

wavelength of the laser decreases, the 266 nm data are

often used to gain aggregation onset (Tagg)

temperatures. However, in this case, the presence of

uridine has a large effect on the intensity of light

scattered at 266 nm as uridine absorbs light of this

wavelength. Therefore, the data acquired at 375 nm are

used to assess onset of aggregation and demonstrates

the advantage of obtaining SLS intensities at multiple

wavelengths. These data are shown for VcCNT in Figure

6 and indicate that aggregation of this protein succeeds

unfolding. Once more, the addition of uridine has a

stabilizing effect on the protein system.

Figure 6: Thermal ramp of 0.5 mg mL-1 VcCNT in the presence

and absence of 1 mM uridine monitored using SLS at 375 nm.

These data demonstrate that stability measurements of

both conformation and aggregation can be obtained for

integral membrane proteins using optical techniques.

The fact that multiple samples can be used

simultaneously in the UNit with optional 375 laser

means that formulation studies (including addition of

interacting ligands) can help to screen optimal

purification and crystallization (Figure 7) conditions for

these proteins.

Sample Tm (CPM

fluorescence, °C) Tagg (SLS375 nm, °C)

NupC pH 7.4 62.6±0.2 64.4±0.2

NupC pH 7.4, uridine

64.4±0.1 68.3±0.9

VcCNT pH 7.4 52.7±0.2 57.4±1.7

VcCNT pH 7.4, uridine

55.5±0.1 64.5±1.1

Table 2: Parameters extracted from the experimental data presented in Figures 4, 5 and 6.

VcCNT

VcCNT + uridine

Application Note

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Figure 7: Membrane protein crystals.

ConclusionsIt is demonstrated here that it is possible to follow the

unfolding transitions of both globular and integral

membrane proteins using CPM dye in the UNit with

optional 375 laser. This instrument facilitates the

comparison of thermal stability measurements obtained

for low volumes (9 L) of the same samples during the

same experiment from intrinsic fluorescence, from an

extrinsic covalent fluorophore and from static light

scattering.

It is well known that polarity-sensitive extrinsic

fluorescent dyes that are commonly used to measure

conformational stability of proteins are ineffective in

the presence of formulations containing detergents.

Furthermore, whilst the use of intrinsic fluorescence to

study unfolding of membrane proteins is particularly

effective for samples that contain significant extra-

membranous domains, it was observed that for the

proteins used here Trp residues were not a useful

probe. CPM, however, allowed clear observation of

unfolding for proteins containing buried Cys residues via

this optical technique even at low concentrations (< 0.5

mg mL-1) of the protein, making it a highly sensitive

method. This method has been seen to work well for

many membrane proteins that are otherwise difficult to

characterize, particularly integral membrane proteins

such as transporters, ion channels and G protein-

coupled receptors.[4,18,19] It is envisioned that the UNit

with optional 375 laser can be used for stability studies

with such proteins.

Extended features of the UNit with optional 375 laser

including the ability to use a third laser, to probe each

well with multiple device settings and to program

individual device settings for each well are

demonstrated with a range of proteins.

References [1] Zhou Y. & Bowie J.U. J. Biol. Chem. (2000) 275, 6975-

6979.

[2] Postis V.L.G. et al., Mol. Membrane Biol. (2008) 25,

617-624.

[3] Yeh A.P. et al., Acta. Cryst. (2006) D62, 451-457.

[4] Alexandrov A.I. et al., Structure (2008) 16, 351-359.

[5] Ayers F.C. et al., Anal. Biochem. (1986) 154, 186-193.

[6] Eilers M. et al., Biophys. J. (2002) 82, 2720-2736.

[7] Kontopidis G. et al., J. Dairy Sci. (2004) 87, 785-796.

[8] Uhrínová S. et al., Biochemistry (2000) 39, 3565-3574.

[9] Loch J. et al., J. Mol. Recognit. (2011) 24, 341-349.

[10] Sakurai K. et al., Biochim. Biophys. Acta (2009) 1790,

527-537.

[11] Burova T.V. et al., Protein Eng. (1998) 11, 1065-1073.

[12] Sakai K. et al., Protein Sci. (2000) 9, 1719-1729.

[13] Johnson Z.L. et al., Nature (2012) 483, 489-493.

[14] Vedadi M. et al., Proc. Natl. Acad. Sci. USA (2006) 103,

15835-15840.

[15] Matulis D. et al., Biochemistry (2005) 44, 5258-5266.

[16] Patching S.G. et al., Org. Biomol. Chem. (2005) 3, 462-

470.

[17] Middleton D.A., Biochem. Soc. Trans. (2007) 35, 985-

990.

[18] Liu W. et al., Biophys. J. (2010) 98, 1539-1548.

[19] Gutierrez H. et al., Structure (2013) 21, 2175-2185.

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