Conformational disorder and dynamics of proteins sensed by ...
Measuring the conformational stability of …...membrane proteins, which include receptors,...
Transcript of Measuring the conformational stability of …...membrane proteins, which include receptors,...
Application Note
1
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
2
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
3
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
4
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
5
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
6
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
7
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
8
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.
Toll-free: (800) 815-6384 Tel: (925) 587-9800 [email protected] unchainedlabs.com