Interaction of Human Chloride Intracellular Channel Protein 1 (CLIC1

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The interaction of human chloride intracellular channel protein 1 (CLIC1) with lipid bilayers: a fluorescence study Joanna E Hare, Sophia C. Goodchild, Samuel N Breit, Paul M. G. Curmi, and Louise Jennifer Brown Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00080 • Publication Date (Web): 14 Jun 2016 Downloaded from http://pubs.acs.org on June 26, 2016

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Biochemistry

The interaction of human chloride intracellular channel protein 1 (CLIC1) with lipid bilayers: a fluorescence study Joanna E. Hare1, Sophia C. Goodchild 2, Samuel N. Breit3, Paul M. G. Curmi4 and Louise J. Brown1* 1

Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.

2

Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds LS29JT, United Kingdom.

3

St Vincent's Centre for Applied Medical Research, St Vincent's Hospital, Sydney, New South Wales 2010, Australia. 4

School of Physics, University of New South Wales, New South Wales 2052, Australia.

Corresponding Author: Louise J. Brown Department of Chemistry and Biomolecular Sciences Macquarie University, Sydney New South Wales 2109, Australia. Telephone: 61 2 9850 8294 Email: [email protected]

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KEYWORDS: CLIC1, FRET spectroscopy, membrane protein, ion channel, metamorphic protein

ABBREVIATIONS: CLIC: chloride intracellular channel protein; FRET: Förster resonance energy transfer,

dansyl

PE:

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-

naphthalenesulfonyl); TM: transmembrane


2

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ABSTRACT

The chloride intracellular channel protein 1 (CLIC1) is very unusual as it adopts a soluble glutathioneS-transferase like canonical fold, but can also auto-insert into lipid bilayers to form an ion channel. The conversion between these forms involves a large, but reversible, structural rearrangement of the CLIC1 module. The only identified environmental triggers controlling the metamorphic transition of CLIC1 are pH and oxidation. Up to now, there is no high resolution structural data available for the CLIC1 integral membrane state, and consequently, limited understanding of how CLIC1 unfolds and refold across the bilayer to form a membrane protein with ion channel activity. Here we show that fluorescence spectroscopy can be used to establish the interaction and position of CLIC1 in a lipid bilayer. Our method employs a fluorescence energy transfer (FRET) approach between CLIC1 and a dansyl-labeled lipid analogue to probe the CLIC1-lipid interface. Under oxidizing conditions, a strong FRET signal between the single tryptophan residue of CLIC1 (Trp35) and the dansyl-lipid analogue was detected. When considering the proportion of CLIC1 interacting with the lipid bilayer, as estimated by fluorescence quenching experiments, the FRET distance between Trp35 and the dansyl moiety on the membrane surface was determined to be ~15 Å. This FRET detected interaction provides direct structural evidence that CLIC1 associates with membranes. The results presented support the current model of an oxidation driven interaction of CLIC1 with lipid bilayers, and also propose a membrane anchoring role for Trp35.


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Although found mainly in the cytosol, members of the chloride intracellular ion channel (CLIC) protein family (CLIC1-6 in vertebrates) are able to interconvert between a soluble, globular state and a membrane-inserted form capable of transporting anions across the lipid bilayer

1-6

. This striking ability

to alternate between drastically different conformations has led to CLICs being described as belonging to a small class of proteins termed ‘metamorphic’ 7. The structural dynamism of metamorphic proteins can make them particularly difficult to study using conventional protein structure determination methods 8. Structural determination of the CLIC protein family poses further obstacles, as resolving the CLIC ion channel structure must also consider protein-lipid interactions.

The soluble forms of the CLIC family proteins are overall well characterized with the first X-ray crystallography structure described in 2001 for CLIC1 (~24 kDa) 9. The crystal structure obtained under reducing conditions shows CLIC1 to be a small globular protein with a GST-like fold possessing a thioredoxin N-domain and a larger all α-helical C-domain 9. Similarly, crystal structures of several other mammalian and lower order CLIC family members share the same monomeric GST-like canonical CLIC fold

5, 10-12

. Interestingly, under oxidizing conditions, a non-covalently linked dimer form of the

CLIC1 member was also created and isolated 3. The crystal structure of this CLIC1 oxidized dimer state showed that the N-domain had undergone a dramatic structural transition from a β-sheet to an all αhelical domain, revealing a hydrophobic surface through which the dimer subunits were non-covalently linked 3. This unique form for CLIC1 suggested that the hydrophobic surface on the oxidized dimer could form a docking interface required for membrane association and subsequent ion channel function. However, the CLICs are fundamentally different to classical ion channel protein families as they notably lack both a membrane-targeting leader sequence and hydrophobic regions typically seen in ion channel proteins 13. Hence, in order to convert between the soluble monomer and the membrane-inserted structure, the CLIC proteins must likely undergo a large-scale conformational change to transverse the membrane and form the pore required for ion conductance. However, a high-resolution integral membrane CLIC structure has yet to be described. ACS Paragon Plus Environment

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In lieu of a high-resolution integral membrane structure, numerous structural and functional biophysical techniques have provided some insight into the membrane-inserted structure and metamorphic transitions of the human CLIC1 protein. Support for an integral membrane CLIC form with a single pass transmembrane (TM) domain near the N-terminus (Cys24-Val46) first came from proteolytic digestion studies

14

; and was later supported by electrophysiological studies where FLAG epitopes,

fused to either the N- or C- terminus of CLIC1, were detected on opposite sides of the membrane

15

.

Fluorescence quenching studies of CLIC1 then showed that Trp35 in the TM region was positioned in a buried environment upon the addition of membranes representing the TM domain region of CLIC1

17-19

16

; a result later supported by peptide studies

. While these structural studies of a membrane state

have started to provide support for an integral membrane form, the electrophysiological characterisation of the individual CLIC channels are also far from complete. To date, the best-studied CLIC channel is the human CLIC1 which has been characterised independently by three research groups

1-4, 9, 15, 20, 21

.

Together, these studies and others have demonstrated that the channel properties of bacteriallyexpressed CLIC1 proteins in artificial lipid bilayers are similar to channels detected in CLIC1transfected whole cells. Likewise, a similar mechanism of membrane integration and pH-dependent conductance was also found for both in vitro and cellular CLIC1 channels, with evidence emerging of an oligomeric state likely to be responsible for the fully functional ion channel (reviewed in 22).

In addition to the oxidation triggered monomer-dimer structural transition identified by crystallography, a pH triggered intermediate state of CLIC1 possessing a solvent-exposed hydrophobic surface has also been identified through acid-induced destabilization measurements

23, 24

. Although performed in the

absence of a membrane mimetic, the authors proposed that the acidic environment encountered by CLIC1 at the surface of membranes would also trigger a similar structural transition, priming the TM region of CLIC1 for insertion into the bilayer. In support of this view are the many functional studies of CLIC members, which also display pH dependence of the ion channel activity. These studies have shown channel activity to be minimal around neutral pH but increasing rapidly as the pH is decreased, ACS Paragon Plus Environment

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or in some studies, also increased

1, 2, 5, 10, 12

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. However, deciphering whether the pH-dependency of the

channel activity is related to the proportion of membrane inserted CLIC1 or a subsequent gating mechanism is difficult as measuring the amount of CLIC1 bound to the membrane is particularly challenging

2, 16, 25

. As a result, we continue to have limited understanding of the connection between

environmental factors that promote interaction with the bilayer and subsequent ion channel activity 26. One current model is that oxidation of CLIC1 in the presence of membranes promotes membrane interaction

16

, possibly through formation of the intermediate ‘half-dimer’ structural state.

Oligomerization then likely follows so as to form the active ion channel

2, 27

. Thus, directly, the redox

environment can change the structure of CLIC1 but is also observed to modify ion channel activity 3. A better understanding of the factors that trigger CLIC1-bilayer association and insertion, as distinct from ion channel gating, is required to build a model of a CLIC1 membrane state.

A combination of fluorescence measurements can offer a reliable and non-invasive approach to studying the interaction and arrangement of proteins within lipid environments (Figure 1). Förster Resonance Energy Transfer (FRET), which measures the proximity of two fluorescent molecules in a donoracceptor pair, is one such biophysical approach. We have previously performed FRET measurements to obtain distances both within, and between, CLIC1 subunits

25, 27

. These measurements revealed intra-

subunit conformational changes and allowed the modeling of a CLIC1 oligomeric state, likely present in the membrane environment

27

. In this present study, we use the FRET approach for the first time to

establish direct structural evidence of the interaction between CLIC1 and the membrane. FRET measurements were obtained between the single native Trp35 residue of CLIC1 to a dansyl label located on the head group of the lipid bilayer. Modified Stern-Volmer acrylamide quenching of Trp35 fluorescence was also performed to assess the CLIC1-membrane interaction and allow for an estimate of the proportion of CLIC1 interacting with the bilayer under our experimental conditions. We describe how these approaches can together be used to begin to map the interaction of CLIC1 with the bilayer.


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This approach is an important step towards obtaining a better understanding of the elusive CLIC membrane state.

EXPERIMENTAL PROCEDURES Expression and purification of CLIC1-WT and CLIC1-W35F Recombinant wild type (WT) CLIC1 (GenBank accession number NP_001279) was expressed in the pET28a vector (Novagen) as a fusion protein with an N-terminal 6x His-tag followed by a thrombin cleavage site as previously described

27

. Briefly, Escherichia coli BL21(DE3) cells were grown in

2 x YT media at 37°C and induced with 1 mM isopropylthio-β-galactoside (IPTG) at mid-log growth phase. The cell culture was grown for a further 16 h, at the reduced temperature of 20°C, before cell lysis. Soluble CLIC1 was purified from the total cellular lysate by binding to Ni-NTA His-Bind® resin (Novagen). CLIC1 was eluted via cleavage of the His-tag following a 16 h incubation with 100 NIH units of bovine plasma thrombin per litre of cell culture (Sigma). Following incubation with 5 mM dithiothreitol (DTT) for 0.5 h, the CLIC1 reduced WT monomer was further isolated by size exclusion chromatography on a Superdex-75 prep grade high performance column (GE Healthcare). The expressed CLIC1 protein consisted of the CLIC1 sequence with an extra three residues at the Nterminus (Gly-Ser-His) as a result of the thrombin cleavage site in the fusion construct. The QuickChange site-directed mutagenesis kit (Stratagene) was used to create a ‘Trpless’ CLIC1-W35F construct for FRET experiments (‘Acceptor only’ sample) by mutating the single native tryptophan to phenylalanine (W35F). The Trpless CLIC1 construct was purified as above for WT.

Liposome vesicles 100 nm liposome vesicles were prepared as previously described

25

. For fluorescence quenching

experiments, soybean phosphatidylcholine (Sigma P5638) and cholesterol (Sigma C8667) were dissolved in chloroform and mixed in a molar ratio of 85:15. For the protein-to-lipid FRET experiments, ACS Paragon Plus Environment

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dansyl-PE

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(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-

naphthalenesulfonyl) Avanti Polar Lipids, 810330) was incorporated into liposomes as the FRET acceptor. A series of acceptor-containing liposomes were formed by adding the dansyl-PE to the chloroform mix at 0.5, 1.0, 1.5, or 2.0% molar percentage of the total lipid. The surface density of dansyl-PE molecules in acceptors per square angstrom (σ) is calculated by assuming that each cholesterol and phospholipid molecule in the cholesterol-phospholipid bilayer occupies 37 and 48 Å2 of surface area, respectively 28. The liposome vesicles were formed by evaporating the chloroform under a stream of nitrogen gas with further drying under vacuum for several hours. Lipids were then rehydrated in assay buffer (150 mM NaCl, 1 mM EDTA, 50 mM Na3PO4, pH 6.0) before extrusion through a 100 nm polycarbonate filter using a LiposoFast Basic apparatus (Avestin). Unilamellar vesicles of 100 nm were confirmed by Transmission Electron Microscope (Philips CM10) and dynamic light scattering with a 633 nm laser source (Malvern Instruments Zetasizer Nano-ZS). No notable changes on the lipid integrity or size of liposomes were observed by TEM following the addition of CLIC1 protein or oxidation (data not shown). Liposome vesicles were used within 24 h of rehydration to ensure lipid integrity was maintained.

Quenching of Trp35 fluorescence with acrylamide Tryptophan quenching by acrylamide was performed on both the soluble form of CLIC1 and upon the addition of phospholipid liposome vesicles. Acrylamide is an effective quenching agent of solvent exposed Trp fluorescence. Fortuitously, CLIC1 possesses a single native tryptophan (Trp35) residue within the TM region. Trp35 of CLIC1 was excited at 290 nm and the observed fluorescence emission (Fobs) recorded at 345 nm. Small aliquots from a 5 M stock solution of ultrapure acrylamide stock solution (Sigma) were added to CLIC1 and CLIC1-liposome suspensions (molar ratio 1:200) for final acrylamide solution concentrations [Q] ranging from 10 to 200 mM. Acrylamide quenching profiles were recorded for reduced CLIC1 monomer (5 mM DTT), both in the presence and absence of


8

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liposomes; and oxidized CLIC1 (addition of H2O2 to 2 mM), also in the presence and absence of liposomes. Absorbance of the liposome vesicles and acrylamide was less than 0.1 cm-1 at the emission wavelength of interest (345 nm). Thus light scattering and inner filter effects were minimal on the fluorescence emission spectra, although still not ignored. All Fobs spectra were therefore corrected for dilution, inner filter effects produced by the light scattering from lipid vesicles, and increasing concentration of acrylamide. The final background-subtracted fluorescence emission (F) was calculated according to: =

29

(Eqn. 1); where ODex and ODem are the absorbance of the sample at 290

nm and 345 nm, respectively.

Quenching data were initially analyzed according to the Stern-Volmer relationship,

= 1 +

(Eqn. 2), where F0 is the corrected fluorescence intensity in the absence of any quenching agent and F is the background-subtracted fluorescence emission as recorded at each increasing concentration of acrylamide quencher [Q]. KSV is the Stern-Volmer quenching constant which reflects the accessibility of Trp35 to acrylamide. However, as acrylamide does not readily partition into a membrane bilayer, the KSV value is considered to best reflect the bimolecular rate constant for dynamic quenching of a Trp residue in the aqueous phase. A Modified Stern-Volmer plot was therefore employed to resolve the ‘accessible’ (a) from the ‘inaccessible’, or buried (b), population of Trp35 residues that arise as a result of interaction with liposome vesicles

30

. In this case, the total fluorescence in the absence of quencher

(F0) is the sum of the fluorescence intensities arising from both the accessible (F0a) and buried (F0b) fractions i.e. = + (Eqn. 3). The modified form of the Stern-Volmer equation: !

"#. %#

=

!

+ " (Eqn. 4) 29, can then be used to determine the fraction of molecules that are accessible (fa) to & #

the quencher agent and the Stern-Volmer quenching constant (Ka) of the accessible population. In this


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present study, the fraction of accessible residues (fa) will be the proportion of CLIC1 in solution or the ‘soluble fraction’.

Fluorescence emission spectra – Trp35 to dansyl-PE Steady-state fluorescence intensity spectra were recorded at 22°C using a Perkin-Elmer LS50B fluorimeter operating in ratio mode with spectral bandwidths of 2.8 - 3.2 nm for maximal fluorescence of the Trp donor. CLIC1-Trp35 to dansyl-PE donor-acceptor pair samples (DA) were excited at a wavelength of 290 nm (λex) and emission (λem) monitored from 300 to 460 nm. The dansyl label on the phospholipids does not contribute to the fluorescence signal over this range. CLIC1 and liposome vesicles were mixed in a 1:200 molar ratio in assay buffer to a final concentration of lipid mixture of 1.6 mM. Each fluorescence spectrum was the accumulation of 4 measurements with a scan rate of 300 nm/min. Four biochemically equivalent samples were prepared in parallel: (i) Blank (B) sample: 8 µM Trpless W35F CLIC1 in the presence of 1.6 mM unlabeled lipid; (ii) Donor only (D) sample: 8 µM WT CLIC1 (Trp35 Donor) in the presence of 1.6 mM unlabeled lipid; (iii) Acceptor only (A) sample: 8 µM Trpless CLIC1-W35F in the presence of labeled lipids (varying percentage incorporation of dansylPE Acceptor - 0.5, 1.0, 1.5 or 2.0%); and (iv) Donor & Acceptor (D+A) sample: 8 µM WT CLIC1 (Trp35 Donor) in the presence of labeled lipids (similarly, varying percentage incorporation of dansylPE Acceptor - 0.5, 1.0, 1.5 or 2.0%). Following the addition of CLIC1, samples were incubated for a minimum of 0.5 h under reducing conditions or 4.0 h for oxidizing conditions, before spectral measurements were taken

27

. All fluorescence emission spectra were corrected for background

fluorescence and scatter by subtraction of the corresponding Blank (B) sample.

FRET - ‘Distance of closest approach’ In general, FRET is a distance-dependent interaction between the electronic excited states of two chromophores in which excitation is transferred from an excited donor (D) molecule (e.g. Trp) to an ACS Paragon Plus Environment

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acceptor (A) molecule (e.g. dansyl) without emission of a photon. The characteristic distance at which the donor fluorescence and FRET are equally probable is defined as Ro i.e. distance between the (D+A) pair that gives a FRET efficiency of 50%. A relative simple application of FRET in membrane systems containing proteins bearing fluorescent residues, such as Trp, is the determination of the transverse location of the latter relative to the bilayer. In our case, Trp35 is the energy transfer donor (D) to dansyl acceptor (A), which is located on the lipid headgroup (dansyl-PE). However, the situation is complex in membranes as each CLIC1-Trp35 donor molecule is likely to be surrounded by an ensemble of dansyl acceptors at non-correlated distances (Figure 1B). Thus, analysis of energy transfer as a single (D+A) distance in this situation is not feasible as the decay of the donor emission is dependent on the topology of the system under study, as well as the concentration of acceptors. In this situation, the use of simplified analytical treatments for the energy transfer efficiency can be applied as a function of Ro and the surface density of acceptors (σ). In such a situation, the FRET efficiency on a surface is determined by the ratio of the donor emission quantum yield in the absence (QD) and presence of the acceptor (QDA) according to ' = 1 − to

&)

&)*

=

)

)* *

&)* 31 . &)

This ratio is calculated from the fluorescence emission spectra according

(Eqn. 5) where FD, FA and FDA are the maximum fluorescence emission intensities

taken at 343 nm. The ‘Distance of closest approach (L)’ between donors in an infinite plane and randomly and uniformly distributed acceptors in an infinite parallel plane is then obtained from &)

&)*

=1+

+,-.

-

0

/ (Eqn. 6)

32, 33

. The slope of the plot of QD/QDA as a function of σRo2 allows for

calculation of L.

For the Trp to dansyl-PE FRET probe pair, Ro is 24 Å

31

. As CLIC1 is added to pre-formed liposome

vesicles, insertion is unidirectional, and due to the effective range of this FRET probe pair, the extent of energy transfer to acceptors at the inner surface of a bilayer of thickness of ~50 Å is negligible; and thus not considered in the calculations. FRET, and consequently L, was also corrected for the fractional labeling of acceptor, typically defined in the literature as ‘fa’ but termed here as ‘facc’. (note, fa is also ACS Paragon Plus Environment

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commonly used in the modified Stern Volmer equation to represent fraction of accessible residues). This term facc., is the population of D molecules that are in close enough proximity to A molecules for energy transfer (E) to occur according to ' = 1 −

)* )

!

"#11.

(Eqn. 7). In our case, facc. represents the

fraction CLIC1 protein that is in close proximity to the dansyl acceptors in the bilayer for FRET to occur, that is, the membrane associated fraction. This fraction can be estimated using 344 = 1 − 3 (Eqn. 8) where fa was obtained from the Stern-Volmer quenching data (Eqn. 4).

To verify our method for establishing interaction of CLIC1 with the membrane, the non-ionic detergent Triton X-100 (Sigma), was added to FRET samples to a final concentration of 0.2% v/v after the final measurement (24 h) was recorded for each dataset. This was to ensure disruption of the (D+A) FRET interaction occurred and resulted in the summative recovery of each Donor and Acceptor equivalent spectrum (data not shown).

RESULTS Oxidation promotes association of CLIC1 with the membrane: Stern-Volmer quenching of Trp35 Acrylamide, a quencher of tryptophan fluorescence, was used to probe the solvent accessibility of the single native Trp35 residue of CLIC1 in the presence and absence of lipid vesicles. Stern–Volmer plots of the quenching of Trp35 were performed under both reducing (5 mM DTT) and oxidizing conditions (2 mM H2O2), as shown in Figure 2. The Trp fluorescence decreased in a concentration-dependent manner upon titration with acrylamide for all samples. Samples where the Trp residue is least accessible to quencher, or within a buried environment, result in the smallest KSV values (Eqn. 2).

The Stern-Volmer plots in Figure 2 show that the Trp35 residue is most accessible to the acrylamide quencher in solution and under reducing conditions KSV = 15.9 M-1, r2 = 0.985. There was no significant change in this observed KSV value upon the addition of liposome vesicles (15.5 M-1, r2 = 0.995). There ACS Paragon Plus Environment

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was therefore little to no change in the local environment surrounding Trp35 upon addition of lipids under reducing conditions. In contrast, the Stern-Volmer quenching constant for CLIC1 under oxidizing conditions in solution was lower than that observed for reducing conditions (KSV = 13.1 M-1, r2 = 0.981). This small decrease in the KSV value upon oxidation of CLIC1 in the absence of lipids may be attributed to the oxidation triggered conformational change where a more buried environment is observed for Trp35 in the dimer crystal structure 3. A considerably lower KSV value of 8.9 M-1 was obtained for CLIC1 under oxidizing conditions in the presence of liposome vesicles. This small KSV value suggests that the Trp35 residue in this lipid environment is notably more buried. However, this data also displayed poor linear correlation (r2 = 0.948) with a notable downward curvature in the Stern-Volmer quenching profile. Hence, data for CLIC1 under oxidizing conditions in the presence of liposomes was analyzed by the Modified Stern-Volmer method.

Trp35 of CLIC1 is in different environments: Modified Stern-Volmer quenching profile The deviation from linearity for the Stern-Volmer plots for CLIC1 under oxidizing conditions in the presence of liposomes suggest the Trp35 residue may not be in a single environment but instead has differing accessibilities according to = + (Eqn. 3). The inaccessible environment detected under these conditions can be assumed to be as a result of the association and/or insertion of CLIC1 into the lipid bilayer, while the accessible environment arises from CLIC1 that remains in solution. In such a case, quenching of two populations of fluorophores can then be analyzed by a modified form of the Stern-Volmer equation (Eqn. 4) to quantify the fraction of fluorophores accessible to the quencher 30.

The plots of the acrylamide quenching data to the Modified Stern-Volmer equation for CLIC1 in the presence of lipids under both reduced and oxidizing environments are shown in Figure 3. Both fits are linear in nature with the y-intercept providing a measure of the fraction of ‘accessible’ residues (fa) or the soluble CLIC1 fraction. For CLIC1 under reducing conditions in the presence of lipids, the yintercept value of 1.05 (fa = 0.95) suggests that only a very small population (~5%), if any, of the Trp35 ACS Paragon Plus Environment

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residue of the CLIC1 protein would be found in an inaccessible or ‘buried’ environment. However, under oxidizing conditions, analysis of the Modified Stern-Volmer plot revealed the presence of a significant proportion of inaccessible Trp35 residues. The intercept of 1.45 (fa. of 0.69) suggests approximately 30% of the CLIC1 protein is in a buried environment; presumably due to interactions with the lipid bilayer.

The close interaction between Trp-35 of CLIC1 and dansyl-labeled lipids: FRET distance of closest approach Our quenching studies support the model that oxidation in the presence of lipid bilayers promotes interaction of CLIC1 with bilayers

16

. Under oxidative conditions, up to 30% of the CLIC1 protein

moves to a buried environment, likely due to association and insertion into the lipid bilayer. Other approaches have also shown that the interaction of CLIC1 to artificial bilayers is also enhanced when the protein is oxidized

3, 10, 12

. However, what cannot be revealed by all these studies is how CLIC1

positions itself with respect to the membrane. Other approaches must therefore be sought.

Förster Resonance Energy Transfer (FRET) is a well-established procedure for measuring distances in biological systems and detecting conformational changes within a distance range of 20 to 100 Å

32, 33

.

Here we have used the FRET technique to seek direct evidence of the interaction between CLIC1 and the membrane. Specifically, we used a simple quantitative FRET-based approach to study the spatial relationship between the single intrinsic tryptophan residue of CLIC1 (Trp35) to a lipid labeled phospholipid (dansyl-PE). The efficiency of energy transfer from the CLIC1-Trp donor to the dansyl acceptor group naturally depends on many factors including the extent of overlap of the Trp fluorescence emission with the absorption of dansyl, the relative orientation of the D and A transition dipoles, and the distance between the D and A labels.

CLIC1 was titrated into liposome vesicles containing varying acceptor concentrations, and thus varying ACS Paragon Plus Environment

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surface density (σ), of dansyl-labeled phospholipids (Figure 4). FRET was detected between the CLIC1Trp35 donor and the dansyl acceptor label as observed by a decrease in the intensity of the Trp donor emission peak (~340 nm) by dansyl, which has an absorption range of 310 to 380 nm. As shown in Figure 4, FRET was observed under both reducing and oxidizing conditions. As expected for CLIC1 associating or incorporating into a bilayer environment, the higher the concentration of dansyl acceptors in liposome vesicles, the greater the amount of energy transfer observed. The extent of energy transfer was greatest upon the addition of H2O2, with up to ~25% energy transfer measured in the presence of 2% dansyl (Figure 4).

The occurrence of protein-to-lipid energy transfer in our experiments is simultaneously simple to detect but complex to analyse. Complexity arises as the detected donor to acceptor energy transfer may be due to the presence of multiple distances and distance distributions resulting from the non-random distribution of the acceptors around each donor label (Figure 1). Several different theoretical FRET models have been developed which are able to consider energy transfer efficiencies in situations where randomly distributed molecules exist 31, 39

31-33, 37-38

. Following previous studies of FRET in two dimensions

, we also sought to obtain an estimate of the distance between our D and A pair by making the

assumption that all membrane associated CLIC1 adopts the same conformation. In doing so, this somewhat simplifies the FRET analysis as all Trp donors are then assumed to be located at the same height above, or below, the membrane, thereby forming a donor plane. The second plane is then formed from the acceptors, which are localized at the aqueous membrane interface via their attachment to the headgroup of the phospholipid molecule. Previously reported quenching studies using brominated lipids indicated that Trp 35 is located within the hydrophobic core of the bilayer close to the membrane surface

16

. We therefore used the observed protein-lipid energy transfer in the present experiments to

determine a location or depth of Trp35 relative the lipid headgroups of the bilayer. Furthermore, by considering the proportion of CLIC1 that interacts with the bilayer, as suggested by our quenching data (Figure 3), and therefore able to contribute to the FRET signal, a lower distance limit on this interaction ACS Paragon Plus Environment

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could then be estimated.

From our quenching results and other studies, it was reasonable to assume that any FRET we observed was due to insertion of CLIC1 into the bilayer. Therefore, to obtain the CLIC1-dansyl distance (L) from the energy transfer observed in Figure 4, we assumed that the CLIC1 donor molecules participating in FRET are associated with lipids and are not in their alternative soluble states. Figure 5 shows the plot of the ratio of the Trp35 fluorescence in the absence of acceptor to presence of acceptor (QD/QDA) as a function of increasing dansyl acceptor labeled lipid surface density (σR02). Values for the QD/QDA ratio were obtained using Eqn. 5 following correction for the proportion of CLIC1 interacting with the lipid bilayer (Eqn. 7), as determined by the modified Stern Volmer analysis (Figure 3). Under reducing conditions, the amount of CLIC1 interacting with lipids (facc) was ~5% and, upon oxidation, increased to 30%. Standard errors shown on Figure 5 were therefore obtained by considering an error of 5% when determining the amount of CLIC1 interacting with the bilayer from the quenching experiments. Likewise, for oxidizing conditions, the FRET was corrected for the 30% proportion of CLIC1 interacting with lipid. Errors shown for oxidizing conditions were calculated by also considering an error in the interacting population of CLIC1 with the bilayer of 5% (range of 25 to 35%).

The amount of energy transfer is observed to be near zero for very low acceptor surface density and increases monotonically with increasing incorporation of acceptor into the bilayer. As QD/QDA was found to be proportional to σR02 over a range of σ values, and as indicated by the linear fits in Figure 5, the approximation of Dewey and Hammes (1980)

33

was then used to calculate L. For comparison,

theoretical plots of QD/QDA vs. σR02 were also calculated and displayed on Figure 5 (dashed lines) for various values of the transverse distance L. For FRET under reducing (circles) and oxidizing conditions (squares), the data in Figure 5 can be accounted for by a straight line in both environments (r2 = 0.827; 0.923, respectively). Analysis of the slopes of the experimental data in relation to the theoretical lines yielded a distance (L) value of 24.8 Å for the CLIC1-Trp35 to dansyl-PE pairs under reducing ACS Paragon Plus Environment

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Biochemistry

conditions with upper and lower values of 24.4 to 25.3 Å. Under oxidizing conditions, the distance obtained was 15.2 Å with lower and upper values of 13.1 to 16.6 Å, respectively. This distance range suggests the Trp35 residue is located in very close vicinity to the lipid headgroups. The results also suggest that the Trp35 residue moves to a position closer to the bilayer surface upon oxidation. However, FRET distances must be interpreted with a degree of caution due to assumptions made during their calculation.

As with any FRET measurement, there are known limitations with assumptions that are made for kappa squared (ĸ2) which describes the rotation freedom of the fluorescent labels. R0 values are calculated by assuming that the transition dipoles of the D and A are dynamically randomized during the excited-state lifetime of D and an accepted value of ĸ2 that is then adopted is 2/3. Further, the contribution from the size of the extrinsic labels themselves can also add uncertainty to the distance L being measured. Assuming a value of 2/3 usually yields distances that differ by < 10% from those determined by crystallography when such comparisons can be made 34. The random orientation of the labeled lipid in the membrane is likely to further reduce uncertainty in R0 due to orientation effects. Lastly, and in the case of energy transfer in membranes, the energy transfer is independent of donor surface density as each donor experiences the same average acceptor surface density. However, this assumes that there is a random distribution of the membrane acceptors in the bilayer. We cannot determine from these current experiments if the lipophilic dansyl acceptors differentially associate with the CLIC1 protein or ‘cluster’ upon oxidation; possibly leading to an increase in energy transfer. As such, crowding effects on FRET are primarily dominated by protein concentration, in particular in studies of real biological membranes, in which the area fraction of protein usually exceeds 20% 41. We therefore kept our donor density low in our reconstituted system so as to avoid such effects that would require a more complex analytical solution to be sought 42.


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DISCUSSION Although the structure of the soluble monomeric globular form of CLIC1 was solved by crystallography now over 15 years ago, and the purified protein shown to self-integrate into lipid bilayers to form an ion channel, little headway has been made into unveiling the structure of an integral membrane form. Efforts to isolate a CLIC1-membrane fraction have largely been unsuccessful due to the dynamic nature of the system. Questions therefore remain about the structural transition that allows CLIC1 to reversibly auto-insert into membranes, with little understanding of the external triggers that may drive this process. Identifying tools that can track the structural transition of the CLIC1 ‘metamorphic’ protein and provide structural information for the membrane ‘end-state’ are thus important to understanding its functional role in cells. However, the choice of available structural tools that can not only identify proteins that display metamorphic behavior, but are also able to then isolate a single state or a specific conformation, if indeed metamorphic, is somewhat limited. Despite being low resolution, fluorescence-based techniques continue to provide the best insight into a CLIC1 membrane state. Our demonstrated direct protein-to-membrane FRET approach is a further useful method to add to this toolbox.

In this study, we observed energy transfer between a single tryptophan in CLIC1 and fluorescentlabeled phospholipids incorporated into membrane vesicles under oxidative conditions. This FRET interaction therefore provides direct structural evidence of the interaction between human CLIC1 protein and the lipid bilayer. The transfer of energy was observed to be dependent on the acceptor surface density of the labeled lipids in the vesicles, from which a distance of ~15 Å was then calculated. The quenching results in the first part of this study, and studies from others

16, 18, 43

, were important in

establishing a position for Trp35 within the hydrophobic region of the membrane and not in a solvent exposed region above the bilayer. The relatively short distance measured from the FRET experiments of ~15 Å therefore indicates that the position for Trp35 is likely close to the lipid surface, conceivably assigning Trp35 as a key anchoring residue for membrane interaction. This location of Trp35 near the lipid surface is not entirely unexpected as tryptophan residues in membrane proteins usually reside near ACS Paragon Plus Environment

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the lipid-water interface

Biochemistry 44

. It is the indole ring of the Trp side chain which has been identified to be

responsible for anchoring and stabilizing several proteins to membrane, and may also assist proteins in partitioning and inserting into bilayers 45, 46. Our FRET distance result is consistent with such a key role for Trp35 in the process of CLIC1 membrane insertion.

When performing FRET with protein systems that interact with membranes, there are often further complexities to consider when compared to soluble systems. Particularly, the lipid bilayer environment imparts some fundamental geometric constraints on a system. Fortuitously, CLIC1 undergoes unidirectional, and likely reversible, insertion into the lipid bilayer

4, 15

. Addition of CLIC1 to the

liposomes after their formation therefore ensures insertion of the protein into the outer leaflet of the bilayer. Further, the effective distance that can be measured by the FRET technique roughly falls between 0.5R0 and 2R0. For our probe pair with an R0 of 24 Å, this translates to a distance range of 12 to 48 Å. The short FRET distance we observed (~15 Å) suggests that there is little to no energy transfer to the acceptor molecule plane on the inside of our vesicles. A final consideration to note when working with the CLIC autoinserting system is that interpretation of any FRET distance between a Trp residue and a labeled lipid requires knowledge of the proportion of the CLIC1 population that associates with the lipid bilayer. Measuring the amount of CLIC1 that is associated with the bilayer is complex and has been found to be dependent on experimental conditions. Not only is the ion channel activity of CLIC1 dependent on external factors such as pH

23

, oxidation

4, 6, 47

and membrane composition 1, but the

fraction of CLIC1 protein associated with lipids is also shown to be pH dependent associated enhanced under oxidative conditions

16

2

with the amount

. Our FRET experiments described here were

therefore performed under the same conditions as our Modified Stern-Volmer experiments where the proportion of CLIC1 interacting with the bilayer under our oxidative conditions was determined to be approximately 30%. Irrespective of these many limitations, and other well accepted FRET limitations 48, the FRET interaction detected between CLIC1 and labeled lipids in this study was clear and provided


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for a direct description of the positioning of a residue within the CLIC1 transmembrane region relative to the membrane bilayer.

In conclusion, we have developed a fluorescence-based approach that can be used to build and test models of the CLIC1 transmembrane form under physiological conditions where both the soluble state and the integral membrane state can be studied simultaneously. Alongside electrophysiology experiments that can tell us about pore lining residues

20

, this combined fluorescence approach can be

used to position residues within the TM region of CLIC1. This approach can also be used to clarify the role that external factors such as oxidation and pH have in the CLIC1 membrane insertion process, as distinct from effects on CLIC1 channel gating. In the absence of high-resolution structural data, future work will utilize other CLIC1 to lipid FRET probe pairs to provide additional distances to continue building a model of a CLIC1 membrane state.

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Biochemistry

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Interaction of Human Chloride Intracellular Channel Protein 1 (CLIC1

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