Impact of Differential Detergent Interactions on Transmembrane Helix

Aug 30, 2016 - (21, 22) Newly synthesized MCP is also incorporated into the membrane, where it is stored before its assembly into new bacteriophage. ...
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Impact of Differential Detergent Interactions on Transmembrane Helix Dimerization Affinities Tabussom Qureshi and Natalie K. Goto* Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5, Canada ABSTRACT: Interactions between transmembrane (TM) helices play a critical role in the fundamental processes required for cells to communicate and exchange materials with their surroundings. Our understanding of the factors that promote TM helix interactions has greatly benefited from our ability to study these interactions in the solution phase through the use of membrane-mimetic micelles. However, less is known about the potential influence of juxtamembrane regions flanking the interacting TM helices that may modulate dimerization affinities, even when the interacting surface itself is not altered. To investigate this question, we used solution NMR to quantitate the dimerization affinity of the major coat protein from the M13 bacteriophage in sodium dodecyl sulfate (SDS), a well-characterized model of a single-spanning self-associating TM protein. Here, we showed that a shorter construct lacking the N-terminal amphipathic helix has a higher dimerization affinity relative to that of the full-length protein, with no change in the helical structure between the monomeric and dimeric states in both cases. Although this translated into a 0.6 kcal/ mol difference in free energy when the SDS solvent was approximated as a continuous phase, there were deviations from this model at high protein to detergent ratios. Instead, the equilibria were better fit to a model that treats the empty micelle as an active participant in the reaction, giving rise to standard free energies of association that were the same for both full-length and TM-segment constructs. According to this model, the higher apparent affinity of the shorter peptide could be completely explained by the enhanced detergent binding by the monomer relative to that bound by the dimer. Therefore, differential detergent binding between the monomeric and dimeric states provides a mechanism by which TM helix interactions can be modulated by noninteracting juxtamembrane regions.

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bacteriophage,20 containing a single TM segment. Approximately 2700 copies of MCP encapsulate the single-stranded bacteriophage genome, and are stably inserted into the bacterial inner membrane during infection.21,22 Newly synthesized MCP is also incorporated into the membrane, where it is stored before its assembly into new bacteriophage. Dimerization via the GXXXG motif has been shown to occur in bacterial membranes23 in an interaction that has also been extensively characterized in detergent micelles.24−27 The ability of sodium dodecyl sulfate (SDS) micelles to preserve these dimers has greatly facilitated these studies, with SDS-PAGE analysis showing a broad band distributed between the monomeric and dimeric states for the wild-type protein, providing a convenient method to identify mutations that alter this equilibrium. More recently, peptide models of the MCP TM segment have been synthesized, facilitating the study of dimerization using fluorescent labels.28 Interestingly, SDS-PAGE analysis of these peptides revealed a greater tendency for dimer formation when compared with that of the full-length protein, suggesting that changes in the MCP sequence outside of the TM segment can alter the affinity of the dimer. The magnitude of this effect

INTRODUCTION A large fraction of eukaryotic membrane proteins are bitopic, with one transmembrane (TM) segment typically acting as a membrane anchor for large interacting extramembraneous domains.1,2 However, interactions also occur between TM segments themselves in a wide range of biological processes, as seen in the activation of receptor tyrosine kinases,3 assembly of cell adhesion receptors,4 and in the formation of ionconducting channels.5 High-resolution structures of these oligomeric states have provided insights into how these associations occur and how they may be regulated or disrupted.6 These structures were often facilitated by the ability of detergent micelles to reconstitute functionally relevant oligomers under conditions that are compatible with solution NMR.7,8 These conditions have also provided a means to measure TM helix association affinities because in many cases distinct peaks can be observed for each oligomeric species in the NMR spectrum and intensities can be quantitated to determine relative populations.9−16 These studies have provided insights into the determinants of both stronger and weaker TM helix interactions, with a significant number of them being nucleated by the GXXXG motif that allows intimate backbone contacts across the interface.17−19 One of the model systems that has been extensively used to characterize GXXXG-mediated dimerization is the 50-residue major coat protein (MCP) from the Ff family of filamentous © 2016 American Chemical Society

Received: July 20, 2016 Accepted: August 18, 2016 Published: August 30, 2016 277

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Table 1. Peptide Sequences

a

Amphipathic helix and TM helix are shaded.31

b15

N-labeled residues are given in bold.

Table 2. Standard Free Energy and PDC Aggregation Numbers for MCP TM Helix Associationa sample

ΔGo (kcal/mol)

e

m

d

f

ΔGomicelle (kcal/mol)

MCP MCPTM MCPTM−M28L/V31L

−2.7 ± 0.1 −3.3 ± 0.1 −3.8 ± 0.1

78.4 ± 0.6 78 ± 1 75 ± 1

79.7 ± 0.5 88 ± 1 89 ± 1

77.2 ± 0.1 80 ± 1 78 ± 1

0.05 ± 0.01 0.23 ± 0.03 0.33 ± 0.03

0.70 ± 0.05 0.6 ± 0.1 0.3 ± 0.1

a Parameters for free energy, PDC aggregation numbers (e, m, and d), and excess detergent release in the dimerization reaction ( f) are defined in the Materials and Methods.

previously described,37 where 15N-labeled valine was added to a final concentration of 100 mg/L. The bacteriophage was isolated as previously described,38 and MCP was extracted from the bacteriophage using phenol extraction.34,39 RP-HPLC was carried out on the phenol fraction with an aqueous 30%−100% (v/v) acetonitrile gradient with 0.01% (v/v) trifluoroacetic acid at a rate of 2% per min on an Agilent C3 semipreparative column. The peak corresponding to the MCP eluted at ∼60% acetonitrile, and the collected fractions were lyophilized and solubilized in aqueous SDS, pH 5.5, just before analysis. A peptide composed of MCP residues 21−48 was synthesized by CPC Scientific (Sunnyvale, CA) with 15Nlabeled valine, glycine, and leucine for a total of 12 labeled amino acids spanning the dimeric interface (MCPTM). Three non-native lysine residues were included at the N-terminus, and the C-terminus was amidated to increase the peptide solubility.40 Protein and peptide concentrations were determined in duplicate using the bicinchoninic acid (BCA) protein assay (Pierce Biotech). Peptide sequences are shown in Table 1. CD Spectroscopy. Far-UV circular dichroism (CD) spectra were recorded on a Jasco-810 instrument with 0.1 mM MCP in SDS, pH 5.5, under the detergent−protein molar ratios that favored either the monomer or the dimer. Eight scans were performed from 250 to 200 nm, with a step resolution of 0.2 nm, a speed of 20 nm/min, a bandwidth of 1.0 nm, and a response time of 2 s, using a 0.1 mm path length quartz cuvette. NMR Spectroscopy. 1H−15N HSQC spectra were acquired for the measurement of monomer and dimer populations on 0.7−1 mM 15N-labeled MCP or 0.5 mM selectively 15N-labeled MCPTM, in 10% D2O with 50−500 mM SDS, pH 5.5, 37 °C using the 500 MHz Varian INOVA spectrometer at the University of Ottawa Faculty of Science NMR Facility. Experiments were typically carried out using 32 transients, 128 increments, a 1H spectral width of 7017.4 Hz, and a 15N spectral width of 1267.2 Hz. An interscan delay of 4 s was used in experiments measuring equilibrium populations of the monomeric and dimeric states. NMRPipe was used to process the spectra and measure peak volumes with its nlinLS module.41 Approximately 0.8 mM 15N-/13C-labeled full-length MCP was dissolved in 100 mM SDS and 10% D2O, pH 5.5, for chemical shift assignment experiments carried out at the Health

has not been measured, nor has its origin been determined. However, it is known that the amphipathic helix interacts with the surface of the micelle, in a nanosecond timescale exchange between the bound and free states,29−33 raising the possibility that this interaction could influence the energetics of TM helix association. To investigate the influence of the amphipathic helix on TM helix association in MCP, we have characterized the secondary structure of the monomeric and dimeric states of full-length MCP and its TM segment alone (MCPTM) in SDS micelles and confirmed that its helical structure is minimally altered by dimerization. We have also used solution NMR to quantitate the energy of this association and found that the standard free energy in 1 M micellar SDS was 0.6 kcal/mol lower for the peptide relative to that of full-length MCP. However, SDS micelles did not act as an ideal solvent when higher protein-todetergent ratios were used, with the affinities being affected by the concentration of empty micelles, suggesting that the empty micelle is an active participant in the monomer−dimer exchange reaction. When standard free energies were calculated with the empty micelle as a participant, the free energy of association between the full-length and TM-peptide MCP was found to be equivalent. This analysis revealed that the increase in apparent affinity that is observed for the TM peptide can be attributed to a larger number of detergent molecules in the protein−detergent complex (PDC) for the monomer relative to that for the dimer or protein-free micelle. Therefore, differences in the detergent-binding properties between the monomeric and dimeric state species, and having no changes in the inherent ability of TM segments to interact, account for the change in the affinity of TM helix self-association for MCP in SDS.

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MATERIALS AND METHODS MCP and MCPTM Sample Preparation. M13 bacteriophage was propagated as previously described34 in Escherichia coli K38A cells (HfC+, T2R, relA1, pit-10, spoT1, ton A22, ompF627, phoA4, T2Rƛ1) 35 in M9 minimal media 36 supplemented with a 1× Gibco MEM vitamin solution, where 10% (w/v) 15NH4Cl and 30% (w/v) 13C-labeled glucose were provided as nitrogen and carbon sources for homogenous 15Nand 13C-labeling. Selectively 15N-valine-labeled bacteriophage was produced in the auxotrophic G11a1 strain of E. coli as 278

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Canada NMR facility on a Bruker Avance III 600 spectrometer. These included HNCACB, CBCA(CO)NH, and 15N-HSQCNOESY (with 100 ms mixing time) spectra. Assignments were made in NMRView.42 Average backbone amide chemical shift differences (Δδ) between the monomeric and dimeric forms of MCP were calculated as Δδ = ((ΔδHN)2 + (ΔδN/5)2)0.5, where ΔδHN and ΔδN are the chemical shift differences between the monomeric and dimeric species for the amide proton and nitrogen atoms, respectively. Diffusion Coefficient Measurements. To measure the diffusion coefficient (Ds) values of selectively 15N-valine-labeled full-length MCP, the bipolar-gradient-pulse-pair longitudinaleddy-current-delay (RAW-BPP-LED) sequence was utilized.43 The length of the position-encoding gradients (δ) was 1.2 ms, and the duration of the diffusion delay (T) was 100 ms. At each gradient strength, 1024 transients were accumulated, with an interscan time delay of 2 s. The fraction ( fz) of the maximal gradient strength (Gmax = 69.9 G cm−1) was increased by 2% increments from 4% to 90%, queued in a randomized sequence to reduce systematic errors. Spectra were analyzed using VNMR (Agilent), the intensities (I) of the resolved peaks were determined by integration, and the standard error was determined from duplicate experiments. Intensities were plotted against the fraction gradient strength, which gives a monoexponential decay that was fit to

[D] [M]2

(1)

[M] [SDS]Micellar

=

Kd +

nM nSDS

2M ⇌ D + Micempty + f Micempty

where f is the fraction of empty micelle aggregation number (e) f=

2m − d − e e

(8)

and m and d are the number of detergent molecules associated with the monomeric and dimeric PDCs, respectively. In the case where the amount of detergent released in the dimerization reaction is less than e, f will be a negative number. According to this model,9 the equilibrium for association is given by Ka =

(3)

[D][Micempty ]1 + f [M]2

(9)

where the concentration of empty micelles is [Micempty ] = [SDS]Micellar − 1/2fd × [MCP] × d − (1 − fd ) × [MCP] × m e

(10)

This gives a free energy of association under standard conditions of 1 M empty micelles (ΔGomicelle) as o ΔGmicelle = ΔGapp − (1 + f )RT ln[Micempty ]

(11)

A plot of ΔGapp versus ln[Micempty], therefore, should give rise to a straight line if the dimerization follows this model.

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(4)

RESULTS AND DISCUSSION NMR of the MCP Monomer−Dimer Equilibrium in SDS. As has previously been observed,15,24,47−49 when MCP is solubilized in SDS, several residues show two peaks in the 1 H−15N HSQC spectrum, with the relative intensity of each peak being affected by the molar ratio of the micellar detergent

where [SDS]Micellar is the concentration of micellar SDS. This allows the calculation of a standard free energy of association under standard conditions of 1 M micellar detergent as ΔGo = ΔGapp − RT ln[SDS]Micellar

Kd +

As has been suggested in the previous models that treated the total micellar detergent as a continuous phase,9,45,46 the amount of detergent that is associated with the monomeric and dimeric PDCs may differ and may also be larger or smaller than the number of detergent molecules contained in an empty micelle. This is recognized in the empty micelle model,9 where the amount of detergent that is released upon dimerization may not be the same as the amount of detergent required to make up the empty micelle, which can be represented by a modified equilibrium

[D][SDS]Micellar [M]

=

2M ⇌ D + Micempty

Standard Free Energy of Association in a Continuous Detergent Phase. The associating polypeptides are confined to the micellar phase which, in previous calculations of TM helix association free energies, has been approximated as a continuous phase.44 According to this model, the equilibrium constant for the association reaction depends on the effective concentration of each species in the micellar phase, giving rise to a detergent-concentration-normalized expression for the equilibrium constant 2

+ [M]

nM nSDS

Standard Free Energy of Association in Micelles. A more recent model for association suggesting that the empty micelle (Micempty) is an active participant in dimer dissociation has been proposed.9 According to this model, the free energy of dimerization would be based on

which can be used to calculate an apparent free energy of association

Ka =

[SDS]Micellar Ka

[M] [SDS]Micellar

(7)

(2)

ΔGapp = −RT ln K app

[M]

fd =

where the reference intensity Io(fo) was measured at fo = 0.02 and the delay following each gradient pulse (τ) was 0.2 ms. Standard Free Energy Calculations. Apparent Free Energy of Association. Concentrations of the monomeric and dimeric species ([M] and [D], respectively) were determined from the peak intensities in 1H−15N HSQC spectra and used to calculate an apparent equilibrium constant for the TM helix association in micelles K app =

(6)

then the relationship between the molar ratio of monomeric MCP to SDS (nM/nSDS) and fd is given by

I(fz ) = Io(fo ) exp[−(γδGmax )2 (fz 2 − fo 2 ) (Δ − δ /3 − τ /2)]

[D] [D] + [M]

fd =

(5)

If the fraction of the dimer population (fd) is 279

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to protein (Figure 1a). These two peaks correspond to a monomeric and a dimeric state, with the monomer being

Figure 2. Chemical shift changes for MCP and MCPTM in SDS. (A) Average backbone 1H−15N amide chemical shift differences (ΔδHN) between the monomer and the dimer for MCP (light bars) and MCPTM (dark bars). (B) Secondary chemical shift differences from random coil values calculated for backbone Cα atoms in MCP plotted as a function of the residue number for the monomer (dark bars) and the dimer (light bars).

Figure 1. Monomer−dimer equilibrium of MCP in SDS monitored by NMR. 1H−15N HSQC spectrum of (A) 0.8 mM MCP in 100 mM SDS and (B) 0.5 mM MCPTM in 50 mM SDS, both in 10% D2O, pH 5.5. Black and red arrows highlight peaks from the monomeric and dimeric states, respectively.

favored as the detergent-to-protein ratio is increased. For this 0.8 mM 15N-/13C-labeled MCP sample solubilized in 100 mM SDS, the peak-intensity ratios of the two species indicate that 42 ± 2% of the population exists in the dimeric state. This sample was used to obtain backbone chemical shifts for both species, building on previous studies on the 15N-labeled MCP that assigned well-resolved doublets for a subset of TM segment residues.24 Average backbone amide chemical shift differences were observed between the monomer and the dimer over the entire length of the TM segment until the second-last residue (Figure 2a), with the exception of Phe45, for which only one peak was observed. Secondary Cα shift analysis showed that the secondary structure of the two states was indistinguishable (Figure 2b), with a helix predicted for residues 6−19 and 26− 45, in line with previous observations on the monomer.24,29,49 This was confirmed by CD spectroscopy on MCP recorded under conditions that favored dimeric states over monomeric states, with superimposable spectra obtained in each case (Figure 3). Therefore, the dimerization of MCP does not give rise to any change in the secondary structure, similar to other self-associating TM helix systems.50,51 Because MCP is restricted to the micellar detergent phase, equilibrium populations of the monomer and the dimer can be altered by changing the molar ratio of micellar SDS to MCP ([SDS]m:[MCP]). Assuming that micelles can be approximated as a continuous detergent phase, free energies of association can

Figure 3. No change in the secondary structure upon dimerization. CD spectra of 100 μM MCP (red) or 70 μM MCPTM (blue) in 100 mM (dark spectra) or 15 mM (light spectra) SDS, pH 5.5, 37 °C.

be calculated relative to a standard state of 1 M micellar SDS (ΔGo).44 For this purpose, peak intensities were measured for well-resolved doublets in the MCP spectrum (i.e., Ile22, Trp26, Met28, Ala35, Val33, Thr36, Thr46, and Lys48) over a range of [SDS]m:[MCP] values, giving rise to a ΔGo of −2.7 kcal/mol (Figure 4). This is a small interaction energy relative to those measured for other TM helix interactions in micelles,51 consistent with our inability to isolate a purely dimeric state under the concentrations that are accessible for this protein in SDS. Comparison of the Dimerization Affinity for FullLength MCP versus MCPTM. To evaluate the impact of the amphipathic helix on MCP dimerization, a similar series of experiments were also performed on a synthetic peptide containing MCP TM segment residues 21−48, with the 15N isotope incorporated into the backbone of glycine, alanine, valine, and leucine (MCPTM). Similar to the full-length MCP, 1 H−15N HSQC spectra of MCPTM showed two peaks for each residue, with relative intensities that depend on [SDS]m: 280

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Figure 4. Association of MCP (blue) or MCPTM (red) in SDS micelles. Each point represents the population of dimer as a percentage of the total protein concentration as determined from the peak intensities measured for each species in the NMR spectrum recorded at 37 °C, with the indicated mole ratio of monomeric protein to micellar detergent. The continuous line represents the calculated distribution for a monomer−dimer equilibrium (eq 7), with a standard free energy of association of −2.7 ± 0.1 kcal/mol (MCP) or −3.3 ± 0.1 kcal/mol (MCPTM).

Figure 5. SDS exhibits nonideal solvent behavior for MCP association. Detergent-normalized association constants for (A) MCP and (B) MCPTM in SDS as a function of the concentration ratio of micellar SDS to protein.

[MCPTM] (Figure 1B). On the basis of the populations determined from the NMR spectra at different [SDS]m: [MCPTM] values, the standard free energy of this interaction was found to be −3.3 kcal/mol (Figure 4), a small but significant increase in the dimerization affinity relative to that of the full-length protein. Its CD spectrum also showed no significant change in the secondary structure under conditions that favored the monomeric or dimeric states, with characteristic spectra for the α-helical structure (Figure 3). Therefore, the enhanced dimerization affinity of the peptide over the fulllength protein cannot be explained by differences in the secondary structure between the monomeric and dimeric states. Our ability to use the measured ΔGo values to quantitate the effect of the removal of amphipathic helix on TM helix affinity relies on the validity of the continuous detergent phase model used to calculate these values. According to this model, the apparent affinity constant (Kapp) will change with [SDS]m: [protein] but can be effectively normalized by multiplying Kapp by the concentration of micellar SDS. However, for both fulllength and TM-peptide MCP, the detergent-normalized Kapp was constant only at higher [SDS]m:[protein] values (Figure 5). At lower mole ratios, the detergent-concentrationnormalized affinity constants increased with decreasing [SDS]m:[protein] values. In spite of the widespread use of the continuum model in the calculation of TM helix association free energies, this analysis suggests that it is not appropriate for the characterization of MCP TM helix interactions in SDS micelles. Potential Causes for the Nonideality of SDS as a Solvent. A potential source of the enhanced dimerization observed for MCP at lower [SDS]m:[protein] values is a change in the size of the PDC. To evaluate this possibility, we measured the translational diffusion coefficients of the PDC by solution NMR using MCP samples that were selectively labeled with 15N-valine, allowing the monomeric and dimeric species for Val33 to be distinguished in a 1D spectrum. Samples were prepared under either low or high [SDS]m:[protein] conditions, and 15N-edited pulse field gradient echo experiments were performed to measure the translational diffusion coefficients.43,52 As shown in Figure 6, at low [SDS]m:[protein] conditions that give rise to deviations from the continuum

Figure 6. Pulsed field gradient-induced decay of normalized MCP peak intensities. (A) Peak intensity ratios for the monomeric state at [SDS]m:[MCP] ratios of 100 (gray) or 300 (black). (B) Comparison of gradient-induced decay for monomeric (black) and dimeric (gray) species measured for [SDS]m:[MCP] = 300. Measurements were made with 15N-labeled valine MCP in 100 mM SDS, pH 5.5, 37 °C. Solid lines are fits to eq 1 using the translational diffusion coefficient of 1.1 ± 0.1 × 10−6 cm2 s−1.

approximation of the SDS solvent, the measured diffusion coefficients were indistinguishable between the monomeric and dimeric states. Moreover, when MCP was diluted to favor the monomeric state, the translational self-diffusion coefficient did not change. Therefore, large changes in the size of the PDC do not appear to account for the observed deviations from the continuum model. 281

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Figure 7. Hypothetical scheme depicting the exchange between the monomeric and dimeric states for TM helices in a micellar system. Light and dark blue bars indicate species that would give rise to peaks in the NMR spectrum that would be assigned as the monomeric and dimeric species, respectively, according to the two different models used for free energy calculations. The continuous solvent model assumes that there is a rapid exchange of peptides between micelles, whereas the empty micelle model would be characterized by slow rates of micelle fission/fusion relative to polypeptide association/dissociation rates. In the empty micelle model, the peak assigned as the dimer would actually be an equilibrium mixture of monomeric and dimeric species.

relationship between ΔGapp and ln[Micempty], where the slope allows determination of the amount of detergent released by the dimerization reaction and the intercept gives the standard free energy of association in a 1 M solution of micelles (ΔGomicelle). As shown in Figure 8, when this analysis was performed for MCP and MCPTM, there was a linear relationship between the

One factor that may help explain the nonideal behavior observed for MCP self-association in SDS is the slow rate of exchange between the monomeric and dimeric states on the NMR timescale. On the basis of the smallest chemical shift difference measured between the two states, the rate constant for exchange must be slower than 25 s−1. To understand the origin of the slow exchange kinetics in this weak interaction, it is useful to consider the mechanism by which TM helices associate and dissociate when encapsulated in micelles (Figure 7). Whereas the series of kinetically resolvable steps that are involved in the dimerization of micelle-solubilized peptides is not known, this mechanism likely involves collisions between micelles, with a fraction of these collisions causing the transfer of single polypeptides between micelles to produce a PDC containing two polypeptides. In the hypothetical scheme shown in Figure 7, this could involve micelle fusion followed by some process that restores the micelle size to its equilibrium state, either in a single micelle fission event or through gradual loss of detergent to the solution and/or other micelles. In cases where micelle fusion gives rise to a PDC containing two polypeptides, dimer formation between the two TM segments could proceed. Although speculative, this scheme highlights two events that could account for the slow (on the NMR timescale) rate of exchange: (i) polypeptide transfer between micelles and (ii) helix association. In the continuum model used to calculate standard free energies, helix association rates are assumed to be much slower than the rates of polypeptide transfer between micelles. However, if this is not the case, the polypeptide transfer may instead be the rate-determining step. According to this scenario, the exchange between the monomeric and dimeric MCP could be fast on the NMR timescale, such that the species that we have assigned as dimeric in the NMR spectrum would actually represent a mixture of monomeric and dimeric states, with the chemical shift appearing at the population-weighted average of the two states. This would give rise to an overestimation of the population of dimeric MCP and therefore overestimate the affinity of self-association. An important consequence of a slow polypeptide transfer between micelles is that distinct peaks for the monomeric state would appear only in the NMR spectrum for PDCs containing a single polypeptide. Although it is not known how the exchange between the monomer- and dimer-containing PDCs actually occurs, it may involve fusion of a PDC containing two polypeptides, with an “empty” micelle that contains no polypeptide. This model was recently proposed by the Arseniev group,9 building on Wyman’s theory of linkages53 tailored to the specific case of interacting micelle-bound proteins.46 According to this model, the affinity of dimerization depends on the concentration of empty micelles, which would become limiting at low detergent-to-protein ratios. This predicts a linear

Figure 8. Association free energies fit the model that treats the empty micelle as a reactant. Apparent free energy of association vs ln[Micempty] (aggregation numbers in Table 2) for MCP (blue) and MCPTM (red).

apparent free energy and ln[Micempty] in both cases. A similar relationship has also been observed for self-association of the TM segment from the fibroblast growth factor receptor 3 (FGFR3) in a 9:1 mixture of DPC:SDS and the vascular endothelium growth factor receptor 2 (VEGFR2) in DPC,9 suggesting that this is a common feature of TM-segment interactions in micelles. Differential Detergent Binding Accounts for Differences in Apparent Dimer Affinities. Because the concentration of empty micelles depends on the number of detergent molecules in each micelle species, it is possible to extract detergent aggregation numbers from this analysis. As shown in Table 2, the number of detergent molecules that make up the PDC for monomeric MCP (m) is approximately the same as for dimeric MCP (d). By contrast, there were a larger number of detergent molecules in the PDC containing a single MCPTM peptide, whereas aggregation numbers for the dimer and empty micelles (e) were found to be similar to those determined for full-length MCP. Consequently, dimer formation by the TM peptide is accompanied by a release of detergent that exceeds the amount required to form one empty micelle. This imbalance in detergent aggregation numbers increases the amount of detergent required to form the monomeric PDC for MCPTM relative to full-length MCP, shifting the equilibrium toward the dimeric state for a given detergent−protein ratio. This appears to completely account for the enhanced affinity seen for 282

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interaction when measured in a series of phosphocholine-based alkyl detergents, with more detergent being released than that needed to form a single protein-free micelle. This was more pronounced as the detergent alkyl chain decreased, with greater excess micelles being released and correspondingly higher dimer affinities being measured in phosphocholine detergents of decreasing alkyl chain length.55 In this series, the change in alkyl chain length changes the micelle size, potentially affecting the region of the peptide that interacts with the detergent headgroup. These interactions could give rise to differences in sizes of monomer- versus dimer-containing PDCs in a way that can disrupt or promote TM helix association.

MCPTM relative to that for the full-length protein because ΔGomicelle is the same as for both full-length and TM peptide systems (Table 2). Therefore, the apparent difference in affinity between the full-length and peptide MCP can be attributed exclusively to differences in the detergent-binding propensities between the two systems. To determine whether this differential detergent-binding behavior is a general characteristic of the MCPTM peptide, we performed the same analysis on data that had been previously acquired on MCPTM with mutations introduced that would increase dimerization affinity (M28L/V31L).15 As shown in Table 2, ΔGomicelle for M28L/V31L is lower than that of the WT MCPTM by ∼0.3 kcal/mol, a difference in free energy similar to the difference determined by the continuum model. Similar detergent-binding parameters were obtained for wild-type and mutant MCPTM peptides, with an excess of detergent being released in the dimerization reaction. This suggests that the regions of the polypeptide outside of the TM segment determine the detergent-binding properties of these complexes. The fact that our data could be fit to a model that treats the empty micelle as a reactant in the equilibrium provides evidence that micelle fusion/fission is rate-determining in the equilibrium that is monitored by NMR. Although the actual rates for SDS micelle fusion/fission in our system are not known, they are predicted to be slow based on the electrostatic repulsion that is anticipated to occur between the SDS headgroups of the colliding micelles. This is substantiated by fluorescence decay rates measured for the dilution of Tritonsolubilized pyrene-labeled triglyceride into SDS,54 which were found to be very slow (e.g., ∼0.01 s−1 in 100 mM NaCl at 23 °C). This is even slower than the macroscopic exchange rates estimated for MCP in SDS micelles, where micelle fusion/ fission rates may have been accelerated by the incorporation of the MCP polypeptide into the micelle. Implications for TM Helix Association. The original purpose of this study was to evaluate the influence of the MCP amphipathic helix on TM helix association. Our results show a clear difference in the apparent affinity between TM and fulllength MCP sequences and showed that this difference can be attributed to protein−detergent interactions that differ between the monomeric and dimeric species that enhance the dimer affinity of the TM peptide. This may reflect the influence of the amphipathic helix on the PDC size, with the interaction between the micelle and amphipathic helix potentially helping to keep the aggregation number of the PDC constant between micelles. However, it is also possible that the three non-native lysine residues added to the N-terminus of the TM peptides to facilitate handling was responsible for the increase in detergent aggregation numbers for the PDCs containing a single peptide. Interactions between sulfate headgroups and amine side chains could stabilize a larger PDC in the monomeric state, with steric effects potentially hindering these interactions in the dimer, thereby reducing its PDC size relative to that of the monomer. Evidence that juxtamembrane regions can influence the apparent affinity of TM helix interactions through differential detergent binding is supported by previous studies examining the amount of detergent released in the self-association of micelle-encapsulated TM segments, although those analyses did not differentiate between empty and protein-bound micelles. In contrast to our results on MCP, self-association of the TM helix of glycophorin A (GpA) in SDS released significantly less detergent than is needed to form a stable protein-free micelle.45 However, the opposite trend was observed for the GpA

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CONCLUSIONS We performed a thermodynamic investigation of MCP TM helix self-association and found that the apparent affinity of the TM segment in SDS micelles is higher when the N-terminal amphipathic helix is replaced by a trilysine tag. This difference in apparent affinity could be explained by differential detergent−protein interactions between PDCs containing one versus two MCP polypeptides. When this was taken into account, it was found that there was no difference in TM helix association energy between the full-length and TM-peptide MCP. Our findings help explain the differences in migration behavior seen for MCP versus the TM peptide in SDS-PAGE gels that reflected the increase in apparent dimerization affinity of the peptide25,40 and also highlight the influence of the micellar state of the detergents used to measure the free energies of interaction.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Jeffrey W. Keillor for allowing us to use his CD spectrometer at the University of Ottawa and to Simon Sauvé and Yves Aubin for acquiring NMR data at the Centre for Biologics Evaluation, Health Canada. This work was supported by an NSERC Discovery Grant to N.K.G.

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ABBREVIATIONS MCP, major coat protein; SDS, sodium dodecyl sulfate; HSQC, heteronuclear single quantum coherence; CD, circular dichroism; TM, transmembrane; NMR, nuclear magnetic resonance; PAGE, polyacrylamide gel electrophoresis

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