Structural Properties of gp41 Fusion Peptide at a ... - ACS Publications

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Structural Properties of gp41 Fusion Peptide at a Model Membrane Interface V. Volkov* and M. Bonn Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany ABSTRACT: We explore the structure and orientation of Nterminal (23 amino acids) of HIV gp41 envelop protein at the interface of a phospholipid monolayer. Using surface specific sum frequency generation, we probe the response of the Amide I vibrational states of the protein, and compare the experimental results to the modeled response of several secondary structures that have previously been reported in literature. To obtain the modeled response, we derive the lineshape expressions under cumulant expansion within the Brownian oscillator model, and express the intensities of the theoretical response according to the components of the Raman tensors and the infrared transition dipole moments of the relevant Amide I modes, under different orientation angles. This approach enables us to identify one plausible secondary structure among those considered, and allows us to determine the orientation angle, under which the protein inserts into the monolayer. We discuss the relevance of our findings toward understanding the functionality of this polypeptide at the membrane interface.

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INTRODUCTION The cell membrane is a semipermeable lipid bilayer constituting the biological barrier separating the interior of a cell from the outside environment. The cell membrane mediates a vast number of biochemical reactions and processes, and its structural robustness is therefore a must. Nonetheless, there are several biological events during which phospholipid membrane integrity is compromised, for instance the initial stages of the fertilization process and upon viral infection. Consider the case of infection with the human immunodeficiency (HIV) retrovirus. The envelope gene of this virus encodes for the single surface spike glycoprotein gp160. Before it settles at the viral envelope, T-lymphocyte FURIN protease cleaves the glycoprotein into two segments of 120 (gp120) and 41 (gp41) kilo Daltons. The cleavage is essential for the success of the transfection process.1 Association with the T-lymphocyte membrane occurs upon binding of gp120 with the receptor of the differentiation cluster (CD4) glycoprotein,2 with a C−X−C chemokine receptor type 4, and with C−C chemokine receptor type 5.3 Before gp120 units can bind to the membrane, a sequence of conformational changes must take place, involving the gp41 segments. Specifically, a prehairpin structure must be formed out of a gp41 triplet, and the N-terminals of gp41 sequences must be inserted into the membrane of T-lymphocyte.4 Consequently, each gp41 triplet thus functions as a bridge, where the triple helical structure is adjacent to the HIV envelope, and the triple stranded half is next to the host cell. The virus achieves moving its membrane toward the membrane of the lymphocyte via folding the stranded region toward to the triple helical part in a hairpin fashion.5 Upon contact with the triple helix, the stranded part winds itself around the folded scaffold of the triple helix to form a compact six-helix bundle.6 This is when it © 2013 American Chemical Society

is anticipated that the two membranes (viral and of lymphocyte) come into a full contact. Merging of the two membranes and the formation of a pore are subsequent key events, where structural and dynamic properties of N-terminals of gp41 are of importance. The exact molecular mechanics of the fusion process is rather complex and has not yet been completely understood. At the same time, the process is obviously effective and reliable. It is apparent that understanding the molecular mechanics of the viral fusion event is of a practical benefit. For example, the inhibition of the gp41 folding process was employed recently, for a development of the first commercial vaccine against viral infection.7 Also, engineered liposomes decorated with viral fusion proteins promise novel approaches in drug delivery.8 Given its key role in the membrane fusion process, the Nterminal of gp41 (23 amino acids long) is called the fusion polypeptide of HIV. The primary sequence of this sequence shows a pattern, which resembles a hydrophobic heptad repeat (HPPHPPP)n: here, H and P represent hydrophobic and polar residues, respectively. The hydrophobic heptad motif is typical for many amphiphilic polypeptides. When folded into a helical secondary structure, the hydrophobic heptad gives rise to an oblique distribution of polar and hydrophobic amino acids along the surface of the fold: this is expected to be necessary for a partial and tilted insertion of the helix into the phospholipid bilayer.9 The HIV fusion polypeptide’s structural properties have been under investigation for past two decades. The results Special Issue: Michael D. Fayer Festschrift Received: June 13, 2013 Revised: August 5, 2013 Published: August 5, 2013 15527

dx.doi.org/10.1021/jp405852r | J. Phys. Chem. B 2013, 117, 15527−15535

The Journal of Physical Chemistry B

Article

the SFG response of the peptide in the Amide I region, and compare the response with theoretical predictions in dependence on the secondary structure, and orientation. The approach has the capacity to address the structure of a partially disordered polypeptide at a membrane interface, obviously. Three distinct secondary structures have been projected for the HIV fusion polypeptide at an interface, following experimental and theoretical efforts in bulk suspensions.10−12 We model the SFG response of these three predicted peptide structures at the lipid monolayer. Using ab initio calculations, we retrieve the frequencies, as well as the x, y, z components (in the molecular frame) of the transition dipole moments (TDMs) and of Raman tensors of the Amide I modes. Subsequently, for each case, we prepare a sequence of orientations by rotating the structures in steps of 10°, about the angle Θ with respect to the normal to the surface, and the angle Ψ of rotation around the main axis of the molecule (see Figure 1). For each orientation, we calculate the intensities of

of Fourier transform infrared (FTIR) studies of fusion polypeptide (fp) in contact with phospholipid membrane fragments,10 NMR spectroscopy in sodium dodecyl sulfate (SDS)11 and in phospholipid micelles12 suggest that under a low concentration (fp:lipid = 1:70), the polypeptide inserts into membrane as a helix. In contrast, at a higher concentration (fp:lipid = 1:10), both FTIR and low-temperature magic angle spinning NMR experiments13,14 show evidence for an assembly of gp41 into amyloid-like structures. Furthermore, the NMR experiments14 have specified that the polypeptides, when frozen in membrane fragments, take predominantly antiparallel βsheet arrangements. In addition, structural variance of the molecule was reported in dependence on the polarity of its environment, lipid charge, and presence of divalent cation.15−29 It is conceivable that this reported structural plasticity is closely related to the reported versatility of the polypeptide: specifically, the gp41 N-terminal has demonstrated both lipidmixing and the hemolytic properties in dependence on its concentration.13,17,18,30 The aforementioned structural studies of the gp41 Nterminal have been carried out in bulk samples of membrane fragments, where variances in local curvature, in local density, and in degree of hydration are possible and likely. As a result, these samples will typically display significant heterogeneity in terms of the relative packing and arrangement of lipid molecules, e.g., displaying local variations in intra- and interdigitations and segregation into crystalline and liquid domains. Given this heterogeneity, one would expect a guest molecule such as gp41 to show a corresponding variance in its behavior and therefore its spectral response in such systems. Moreover, in systems containing bulk membrane fragments, it is impossible to address molecular trafficking and binding to the interface as in vivo: the membrane next to the aqueous subphase does not represent well the reticular interface with cellular cytoplasm. As a result, a guest molecule cannot diffuse freely toward to a membrane from an aqueous pool and insert according to its surface activity. Partitioning properties are affected by dehydration and structural heterogeneity. Here, we address protein structural properties specifically at a well-defined lipid interface using sum frequency generation (SFG) spectroscopy. SFG is a second-order nonlinear optical process in which two light beams at frequencies ω1 and ω2 generate light at their sum frequency (ω3 = ω1 + ω2). The process of SFG is highly surface-specific, as the SFG process is forbidden in centrosymmetric media (within the electric dipole approximation). Hence, for centrosymmetric media like liquid water, SFG possesses monolayer interfacial specificity. Typically, surface SFG experiments employ a pulsed visible laser beam of fixed wavelength and a pulsed tunable or broadband infrared laser beam. The sum-frequency signal is enhanced when the infrared frequency matches a vibrational resonance of some interfacial species. Thus, by recording the SFG signal as a function of the infrared frequency, one can obtain an SFG spectrum that contains unique information on the vibrational response of specifically the surface molecules, avoiding the response from the underlying bulk. SFG experiment on lipid monolayer, Langmuir−Blodgett films allow for detailed control over the relevant physical and chemical properties at the interface. Indeed, SFG has been widely used to address molecular structural properties at interface of model phospholipid membrane systems.31−43 We explore the structural properties of HIV fusion polypeptide as it binds to phospholipids at the water−lipid interface. We detect

Figure 1. Orientation model, where Θ, Ψ, and Φ are the Euler transformation angles.

the signals under different polarization conditions. In particular, we follow the procedure suggested by Maker:44 we extract the components of the rotation matrices which project the relevant derived molecular properties onto the axes of the laboratory frame according to the polarization condition. Finally, we average the orientation over angle Φ (as shown in the Figure 1) to account for the invariance of molecular distribution under “in-plane” rotation around surface normal. Finally, we derive the expressions for the line-shapes of SFG response using cummulant expansion technique.45,46 Consequently, we generate a series of spectra (under different polarization conditions) in dependence of Θ and Ψ rotational angles for three representative secondary structures to compare with the experimental data. In this manner, we narrow the conformational space of secondary structural expressions, and extract the most likely molecular orientation at the interface.

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METHODS We obtain SFG spectra of N-terminal of gp41 at the water− lipid interface using a broad-bandwidth SFG spectrometer as described previously.47 Briefly, for the SFG process, we employ a visible (Vis) and an infrared (IR) beam centered at 800 nm and 1675 cm−1, respectively. The energy and the spectral 15528



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bandwidths of the visible radiation are 20 − 30 μJ and 16 cm−1. The energy of the IR pulses is 2 −3 μJ, and its spectral bandwidth is 150 cm−1. Both beams are focused down to 100 μm beam waist (full width at half-maximum, Gaussian profile) and overlapped at the sample surface. To constrain the structural refinement, we conduct two sets of experiments: with the incident angles (relative to the surface normal) of the Vis and IR beams at 35° and 40°, respectively; and with incident angles of 31° and 35°, respectively. The SFG light generated by the sample is detected with a monochromator connected to a charge-coupled device (CCD) camera. Spectral analysis included background subtraction and division by the reference signal from z-cut quartz that is recorded immediately before or after the spectrum of the monolayer. We detect SFG spectra with SSP and PPP polarization setting, where the first, the second, and the third letters indicate polarizations of the SFG, of the visible, and of the infrared radiations, respectively. Zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, 850355), and negatively charged 1,2-dipalmitoyl-snglycero-3-phospho-rac-1-Glycerol (DPPG, 840455) are obtained from Avanti Polar Lipids, Inc. Alabaster, AL. HIV-1 envelope N-terminal of gp41 protein H-Ala-Val-Gly-Ile-GlyAla-Leu-Phe-Leu-Gly-Phe-Leu-Gly-Ala-Ala-Gly-Ser-Thr-MetGly-Ala-Arg-Ser-NH2 (H-2978) is purchased from Bachem AG, Bubendorf, Switzerland. The polypeptide is dissolved in 0.1 mM Phosphate buffer, and injected into the aqueous subphase after deposition of the phospholipid monolayer. The subphase consists of the same 0.1 mM Phosphate buffer. The samples are stable for 1 hour, at least. In all preparations we use ultrapure Millipore water of 18 MΩ·cm resistivity. The lipid monolayer is prepared on a phosphate buffer solution in a homemade trough. We use a tensiometer (Kibron Inc., Finland) to measure the build-up of the surface pressure upon deposition of phospholipid. The surface pressure of phospholipid monolayer is raised sequentially up to 25 mN/m by spreading droplets of 1 mg/mL (phospholipid/chloroform) solution. The surface pressure for each system is calibrated by setting the value of the pure buffer surface to 0 mN/m before spreading the monolayer. We obtain the frequencies and the components of the transition dipole moments and of the Raman tensors of normal modes through ab initio calculations using Gaussian09.48 Specifically, we employ rb3lyp/6-31g* level of theory under options Freq = (Raman) iop(7/33 = 1). The scaling factor for the normal-mode frequencies is 0.95. We optimize three structural realizations of the of HIV gp41 N-terminal according to the protein database (PDB) entries 2PJV, 1P5A, and 2ARI. These structures were concluded from, respectively, FTIR studies in membrane fragments with classical annealing in vacuum,10 NMR response in phospholipid micelles,12 and NMR response in surfactant micelles.11 Figure 2 shows the dihedral angle setting for the selected and optimized structures: the extent of the helical structural component increases in the order of presentation of the three structures.

Figure 2. (A) Dihedral angles (not to be confused with Euler angles) along the polypeptide backbone for the structures as reported in the references 10, 12, and 11, respectively. Green rectangles indicate the extent of the helical components for each of the three structures. Dashed vertical lines track representative numerical values for the dihedral angles of in a typical α-helical protein.

Figure 3. (A) SFG spectra of HIV N-terminal gp41 fusion polypeptide at the interface of DPPC/DPPG Langmuir−Blodgett monolayer detected under SSP (black lines) and PPP (purple lines) polarizations as indicated, with incident angles (relative to the surface normal) of the Vis and IR beams of 35° and 40°, respectively. (B) SFG spectra when the incident angles are 31° and 35°. The lower six panels represent theoretical spectra for the secondary structure reported in the ref 10 under the indicated orientation angles. Left and the right columns correspond to calculations for the experimental geometries employed to detect signals reported in the panels A and B, respectively. Selection of the orientation angles are according to the results reported in the Figure 4.

as may be expected for a complex polypeptide such as gp41, the SFG spectra contain sufficient detail to allow a comparison between the different structures, as explained below. Specifically, a complex substructure is evident: there are numerous states besides the dominant spectral signature. Moreover, there is a noncoincidence of the main spectral signature: it peaks at 1660 cm−1 and 1667 cm−1 for SSP and PPP polarization conditions, respectively. The relative intensities of the two signals indicate that the helical motive, while dominating, is not present in the entire molecule.49,50 Given the predominance of helical motive in the secondary protein structure for relatively

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RESULTS Figure 3 represents the SFG spectra detected under SSP and PPP polarization conditions for the sample where lipid to polypeptide ratio is approximately 70:1. The spectra show intensity in the spectral region characteristic of a predominantly helical protein, consistent with the conclusions from FTIR and NMR studies.10−12 While the overall features are quite broad, 15529



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low polypeptide concentration (polypeptide:lipid =1:70),10−12 we focus in our efforts on SSP and PPP signals, to which we know the helical structure contributes greatly. For higher peptide concentrations, it has been suggested that cooperativity of interactions of the fusion polypeptides plays an important role in the pore formation process, which is expected to contain significant β-type secondary structures. For these studies in particular, PSP polarization experiments would be very useful, but these are topic of future studies. Here, we set out to address structural properties of the polypeptide during the very initial stages of viral infection; Probing helical structures provides an excellent opportunity to do so. To model the SFG response, we consider the analytical expression of the signal according to ref 51: 3 2

I(ω) =

correlation part from the evolution operators’ correlation (addressed in the next section) according to the Born− Oppenheimer approximation. Further, we employ the identities derived analogously to those by Mukamel [Appendix 7A of ref 45] and Cho [page 37, ref 46]: ei / ℏHeτ3e−i / ℏHgτ3 = eiωegτ3 exp−[i

(2) χeff, = L YY (ω)L YY (ω1)LZZ (ω2)χYYZ , q R̃ q(ω) qSSP

ei / ℏHvτ2e−i / ℏHeτ2 = eiωvg τ2 exp−[i

∫0

(5) τ2

e−i / ℏHgτ2 exp+[−i

W (τ′) dτ′]ei / ℏHgτ2e−iωegτ2

∫0

τ2

U (τ′) dτ′]

(6)

ei / ℏHgτ1e−i / ℏHvτ1 = ei / ℏHgτ1e−iωvgτ1e−i / ℏHgτ1

(1)

exp+[−i

⎛ i ⎞2 R SFG(τ1 , τ2 , τ3) = ⎜ ⎟ ⟨μ3̂ (τ1)μ2̂ (τ2)μ1̂ (τ3)⟩ ⎝ℏ⎠

exp+[−i −

(2)

∫0

exp−[i

∫0 τ1

∫0

τ1

τ1

W (τ′) dτ′]

τ3

∫0

W (τ′) dτ′] = 1 − i

dτ

=1+i

(7)

∫0

τ1

W (τ′) dτ′

τ

⟨W (τ′)W (τ )⟩ dτ′

(8)

U (τ′) dτ′]

∫0

τ3

U (τ′) dτ′ −

∫0

τ3

dτ

∫0

τ

⟨U (τ )U (τ′)⟩ dτ′

Opening brackets and sorting the time-dependent terms up to the second order, leads to R SFG(τ1 , τ2 , τ3) =

⎛ i ⎞2 −iωvg τ1 iωvg τ2 ⎜ ⎟ ⟨μ ̂ (τ )μ ̂ (τ )μ ̂ (τ )⟩e e ⎝ℏ⎠ 3 1 2 2 1 3 e−iωegτ2eiωegτ3G(τ1 , τ2 , τ3)

(9)

where G(τ1 , τ2 , τ3) = exp[− −

(3)

∫0

∫0

expressed through the transition dipole moment operators. In Hilbert space this three-point correlation reads:

+

⎛ i ⎞2 R SFG(τ1 , τ2 , τ3) = ⎜ ⎟ ⟨ei / ℏHgτ1μ3̂ (τ1)e−i / ℏHvτ1ei / ℏHvτ2μ2̂ (τ2) ⎝ℏ⎠ e−i / ℏHeτ2ei / ℏHeτ3μ1̂ (τ3)e−i / ℏHgτ3⟩ =

∫0

U and W represent the e−g and v−g frequency gap operators. We may expand the time ordered exponentials up to the second order, for example:

here, LYY(Ω) and LZZ(Ω) are the Fresnel factors for Y and Z components (in the laboratory frame) of the field oscillating at frequency Ω.51 χYYZ,q is the amplitude of the YYZ component of the χIJK macroscopic susceptibility according to spatially averaged molecular properties and the polarization conditions in the experiment. R̃ q(ω) is the line-shape corresponding to the Fourier transform of the time-domain response function. In the following, we provide a theoretical formulation for the response function and its intensity (macroscopic susceptibility). SFG χ(2) Response Function: Brownian Model: Cumulant Expansion. The Dyson theory on the interaction picture time-dependent perturbative expansion52 provides the necessary ingredients to calculate the line shapes of the observed resonances. Following,45,46 we write the three-point correlation function characteristic to χ(2) eff of SFG process:

⎛ i ⎞2 ⎜ ⎟ ⎝ℏ⎠

+

dτ

∫0

τ1

dτ

τ2

τ3

τ1

dτ

∫0

dτ

∫0

τ

⟨W (τ )W (τ′)⟩ dτ′

∫0

⟨W (τ′)W (τ )⟩ dτ′ −

τ2

τ2

∫0

τ3

dτ

∫0

⟨W (τ )W (z)⟩ dz −

⟨W (τ )U (z)⟩ dz + τ2

∫0

τ

⟨U (τ )U (τ′)⟩ dτ′ −

∫0

∫0

τ2

∫0

τ

∫0

∫0

⟨μ3̂ (τ1)μ2̂ (τ2)μ1̂ (τ3)⟩⟨ei / ℏHgτ1e−i / ℏHvτ1ei / ℏHvτ2e−i / ℏHeτ2ei / ℏHeτ3 ⟩

U (τ′) dτ′]

ei / ℏHgτ3e−i / ℏHgτ3

where ni(Ω) is the refractive index of the media i (i = 1 means air) at the frequency Ω; ω1, ω2, and ω are the frequencies of the visible, infrared and SFG radiations, respectively; β is the angle of the SFG wave vector to the normal to the plane of the sample; I1 and I2 are the intensities of the visible, and the infrared radiation fields. χ(2) eff,q is the effective susceptibility of normal mode q. The square over the absolute value of the sum is according to the homodyne character of signals we detect in this work. For a signal under SSP polarization conditions:

e

τ3

2

8π ω Sec [β ] |∑ χ (2) |2 I1(ω1)I2(ω2) c 3n1(ω)n1(ω1)n1(ω2) q eff, q

−i / ℏHg τ3

∫0

∫0

τ1

dτ

⟨W (τ )U (z)⟩ dz −

⟨W (τ )U (z)⟩ dz +

∫0

τ2

dτ

τ3

∫0

∫0

dτ

τ

⟨U (τ′)U (τ )⟩ dτ′

∫0

∫0

τ2

τ3

τ1

dτ

⟨W (τ )U (z)⟩ dz τ2

dτ

⟨U (τ )U (z)⟩ dz]

(4)

(10)

Here, indices g, e and v represent properties of the ground state, of the first vibrationally excited state, and of the virtual electronic state (vacuum interaction) “through” which the SFG light is emitted, respectively. We separate the rotation

The first four ordered integrals are the autocorrelation functions of the e−g and v−g gaps. The others have to be addressed using factorization suggested by Mukamel [eq 8.A.6, ref 45]: 15530



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Figure 4. Numerical difference between the calculated and the experimental SFG spectra in dependence on Euler Θ and Ψ angles, as shown in Figure 1. The blue and green color gradients show the results for SSP and PPP polarization conditions, respectively, where color brightness scales with agreement between calculation and experiment. Panels A, B, and C represent the ab initio results for the structures reported in refs 10, 12, and 11, respectively, when the angles of incidence of the Vis and IR radiations are 35 and 40 degrees. Panels D, E, and F represent the results with incident angles of 31 and 35 degrees. Red circles indicate angular regions for the calculated spectral responses as in Figure 3.

G(τ1 , τ2 , τ3) = exp[−w(τ1) − w*(τ2) − u(τ2) − u*(τ3)

R SFG(t1) =

+ w(τ1) − w(τ1 − τ2) + w*(τ2) − v(τ1) + v(τ1 − τ2)

R(t1) = e−iωegt1 exp[−u(t1)]

− v(τ2 − τ2) + v*(τ2) − v(τ2) + v(τ2 − τ3) − v*(τ3)

∫0

v (t ) =

∫0

u(t ) =

∫0

t

dτ

∫0

dτ

∫0

dτ

∫0

t

t

τ

dτ′⟨W (τ )W (τ′)⟩

τ

dτ′⟨W (τ )U (τ′)⟩ τ

dτ′⟨U (τ )U (τ′)⟩

(12)

Under τ1 = t1 + t2, τ2 = t1, and τ3 = 0, the response function for SFG is R SFG(t1 , t 2) =

⎛ i ⎞2 −iωvg (t1+ t 2) ⎜ ⎟ ⟨μ ̂ (t + t )μ ̂ (t )μ ̂ (0)⟩e 2 2 1 1 ⎝ℏ⎠ 3 1

eiωvet1 × exp[−u(t1) − w(t 2) + v(t1) + v(t 2) − v(t1 + t 2)]

(15)

and the term ⟨μ̂3(t1)μ̂2(t1)μ̂1(0)⟩ represents the macroscopic susceptibility χIJK (capital indices are of the lab-frame),44,56 which is according to rotational average of ⟨αij(t1)μk(0)⟩ function (i, j, k are the indices are of the molecular frame). Here, the two operators μ̂3(t1) and μ̂2(t1) merge to give the expression of the Raman tensor component αij. This is consistent with the definitions in the classical limit.53−55 Macroscopic Susceptibility. In general, signals under SSP and PPP polarizations contain, respectively, contributions from (XXZ, YYZ) and (XXZ, XZX, ZXX, YYZ, YZY, ZYY, and ZZZ) components of the macroscopic susceptibilities χ; and the indices X, Y, and Z are the axes of the laboratory frame. However, given the rotational invariance (with respect to rotation around the surface normal), for the mentioned polarizations, the effective susceptibilities χ(2) eff,q of a normal mode q are determined only by the tensor elements χYYZ, χZXX, χZZZ, χXXZ, and χXZX, as described in ref 51. We calculate the macroscopic susceptibilities using Euler transformations of the components of the Raman tensor and transition dipole moment of the qth normal mode according to44,56

(11)

where, w(t ) =

(14)

where

− v*(τ2) + v(τ1) − v(τ1 − τ3) + v*(τ3)v(τ2) + u(τ2) − u(τ2 − τ3) + u*(τ3)]

⎛ i ⎞2 ⎜ ⎟ ⟨μ ̂ (t )μ ̂ (t )μ ̂ (0)R(t )⟩ 1 ⎝ℏ⎠ 3 1 2 1 1

(13)

It is reasonable, in the case of Raman-type emission, to consider t2 to be negligibly small. Hence, the expression of the timedomain response function (equivalent to the effective susceptibility of SFG process, χ(2) eff obtains the following form:

(2) χIJK = ,q

∑ ijk = xyz

15531

NS⟨ NIi NJj NKk ⟩

∂αij ∂μk ∂Q q ∂Q q

([16])



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Here, index k accounts for the x, y, and z components of the transition dipole moment of normal mode q in the molecular frame. These components may contribute to a response upon action of the operator μ1(τ1) as in eq 3. Analogously, indices i and j run over the x, y, and z components of the Raman tensor αij in the molecular frame. These address properties of μ3(τ1) and μ2(τ2) operators, respectively. The angular bracket term represents spatial averaging of the product of the elements of the Euler matrices, which project the x, y, and z properties derived in the molecular frame onto the laboratory X, Y, and Z coordinates according to the polarization condition in experiment. In our studies, we calculate the components of the transition dipole moment and of the Raman tensor of the Amide I modes using three different initial structures of N-terminal of gp41 polypeptides.10−12 We calculate the SFG intensities of the normal modes (given our specific polarization and geometry settings) by numerically averaging the different orientations using Mathematica (Wolfram). Our approach is equivalent to that presented in ref 56, where analytical expressions are employed. Accordingly, we simulate the pairs of SSP and PPP spectra in the [Θ,Ψ] rotation angular space (Figure 1 defines these Euler angles) gridded by 10 degrees. Each spectrum (or, more specifically, calculated SSP and PPP pair spectra) is normalized by the maximum value of the SSP response. Analogously, the experimental SSP and PPP spectra are also normalized to the maximum value of the experimental SSP intensity. We take the numerical difference between the calculated and the experimental responses to pinpoint the plausible secondary structure upon reaching a numerical minimum. Notwithstanding the low symmetry of the molecule, the SFG spectra calculated for different orientation angles demonstrate a significant variance. This allows us to reach definitive conclusions concerning which of the three proposed structures is most likely: by comparing the experimental spectra with the calculated ones, despite the relatively featureless and broad characteristics of the observed SFG resonances.

analysis presented here, this structure is clearly inconsistent with the experimental data. In particular, it is evident from B that this secondary structure does not provide a consistent reproduction of the experimental results simultaneously under PPP and SSP polarization conditions in the Euler Θ/Ψ orientation space. Panel E shows that, under the experimental conditions relevant to that graph, there is some consistency for Θ and Ψ pair of [110°, 0°] with both PPP and SSP signals. However, since this only constitutes partial agreement, we disregard this secondary structure. Finally, Figure 4C,F clearly shows that the most ordered helical motive, as inferred from NMR experiments on surfactant micelles,10 shows the least consistency with the experimental results. In neither of the two experimental geometries can we find orientation angles so that the calculated PPP and SSP spectra would be close to the detected ones: the blue and the green areas (of the minimal numerical difference) do not coincide in either of the two panels. The results of our SFG spectroscopic study of gp41 Nterminal associated with a lipid monolayer thus agree with the secondary structure anticipated by L. Gordon, et.al.,10 who studied the molecule in bulk suspensions of phospholipid membrane fragments. According to this structure, less than half of the amino acids contribute to the helical motive. This is in agreement with the observation that that the signal under PPP polarization is of similar intensity is that recorded with SSP, as was discussed in previous SFG studies of helical polypeptides.49,50 It is interesting that our results do not agree with the more helically ordered structural realization as anticipated in micelles.11,12 This may be due to the fact that highly curved media may organize the guest molecule’s structural motives in such a way that the length of the guest hydrophobic region matches that of the hydrophobic core micelles. In addition to confirming the secondary structure reported in ref 10, we also infer the orientation of the polypeptide relative to the lipid monolayer: we predict that the angle of insertion of the gp41 N-terminal into the interface of Langmuir−Blodgett is approaching 90 degrees with respect to the surface normal. On one hand, this is not consistent with the 40 degree angle anticipated using polarization-resolved ATR FTIR spectroscopy,21 and ESR studies30 in membrane fragments suspensions. On the other hand, it is not completely obvious that we can directly compare the two results, given the different nature of the model membrane used in the experiments. Arguably, the interaction of the polypeptide with the Langmuir−Blodgett interface resembles more closely the situation in vivo; moreover, the SFG experiments reported here specifically probe the interface due to the nature of second order susceptibility and therefore contributions from polypeptides in the bulk are suppressed. Our results seem to make the connection to the observation in the NMR experiments14 that the gp41 N-terminal polypeptide may take predominantly antiparallel β-sheet arrangements when frozen in phospholipid membrane fragments. Indeed, the near-horizontal character of partitioning at the interface inferred here provides better opportunity for the formation of parallel (or antiparallel) aggregates. Furthermore, panels A and D and Figure 3 suggest that a possible variance in Θ from 80 to 110 degrees may be correlated with the variance of the angle Ψ from 50 to 130 degrees. Such an arrangement is favorable for a relatively axial arrangement and alignment of the side groups. In our analysis we also extract the possible range of angular values for Ψ. To the best of our knowledge, there is no obvious

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DISCUSSION Figure 4 summarizes the spectrally integrated numerical difference between the calculated and experimental spectra, for various combinations of angles [Θ,Ψ]. Panels A and D were calculated starting with the secondary structure reported in ref 10 obtained by combining FTIR spectroscopy of gp41 Nterminal in phospholipid membrane fragments with MD annealing. The panels, for which color intensity reflects the degree of agreement between theory and experiment, indicate that it is possible to find a consistent convergence with both PPP and SSP responses in the central area of the Euler Θ/Ψ orientation space. The lower panels in Figure 3 show a comparison of a series of the calculated spectra (for the Euler angles, as indicated) for the two sets of incident angles in our experiment: left and right sides in the figure. One may clearly see that the calculated spectra for Θ and Ψ pairs of [80°, 50°] and [90°, 90°] reproduce the spectral properties and the intensities of the detected responses fairly well. These spectra, calculated for several discrete angles help to anticipate orientation variances, which are naturally and inevitably present in the sample. Panels B and E (Figure 4) represent the results of the corresponding orientation search for the different secondary structure of gp41 as inferred in ref 12 from studies on phospholipid micelles. Within the framework of a theoretical 15532



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gp41 peptides at the membrane interface is relatively homogeneous.

reference in the literature on this axial orientation of the molecule. Even though the affinity of N-terminal gp41 polypeptide to DMPC is limited, we may adopt the prediction of MD simulations reported in ref 57. Using the reported dihedral angles and the relative position of the side groups with respect to the phosphate of the membrane (Figure 3 in the reference) we infer that Ψ = 70°. This is in the middle of the angular range we conclude here and which was employed in the calculation of the spectra in Figure 3. Anticipation of the Euler Ψ angle in our analysis is possible without any isotope labeling as the system approaches C1-symmetry. As an approach, this should be valid even in structurally symmetric molecules as long as there is a variance in nature of Amide I modes expressed on different sides of the motive of the secondary structure. In fact, recently the variance in the nature of Amide I vibrations was anticipated quantum mechanically even for gramicidin,58 which is considered to show a uniform helical motive. The results reported here demonstrate clearly that SFG spectroscopy not just simply requires a theoretical analysis of much higher intricacy than that suggested three decades ago,59,60 but accordingly offers the opportunity to test the presence of fine structural aspects at the interface. Following the methodology we developed, it is trivial and straightforward to approach further optimization of the experimental spectral properties assuming either coexistence of (at least) two orientations or two secondary structures. We believe, at present this could be more speculative rather than helpful. At present, we consider the ultimate structural analysis at the interface requires a convergence starting from: (i) an extraction of secondary structural solutions anticipated from molecular dynamic predictions through rapid ergodic sampling techniques (parallel tempering and/or metadynamics); (ii) a detection of signals under many different polarization and angular settings. The beauty of the even order spectroscopies at the interface is through the granted diversity of the microscopic susceptibility components due to the limited orientation averaging, as present at the interface. Finally, in our discussion, we wish to address the dynamics through the apparent widths of the detected spectral lineshapes. Strictly speaking, to distinguish homogeneous (dynamic) and inhomogeneous (quasi-static) contributions to the line width, higher order spectroscopies (χ(3) for bulk samples and χ(4) for interfacial spectroscopy) are necessary. However, it is evident that the linewidths inferred from our χ(2)-experiments constitute an upper limit for the intrinsic (homogeneous) linewidths of the Amide I modes. Comparing our inferred Amide I spectral linewidths to the homogeneous linewidths found for a CD3χ helix in bulk membrane environment (measured in a third-order experiment),61 it is remarkable to note that the linewidths reported here for gp41 are substantially (1.5 times, at least) narrower than those reported for the CD3χ helix, despite the additional inhomogeneous broadening that is expected for gp41. Hence, the line-shapes of the Amide I resonances we observe at the interface are narrower than both those observed for CD3χ helix in membrane and that of NMA in aqueous environment.62 We tentatively attribute the observed spectral narrowing to a decreased exposure of the local Amide I sites to water. Since different then in the bulk experiments,61,62 this may be due the dominance of the dry Amide I modes at the hydrophobic side in the detected response. In any case, the relatively narrow spectral features observed here indicate that the organization of an ensemble of

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CONCLUSIONS We employ label-free SFG spectroscopy to characterize the vibrational states of the polypeptide gp41 upon its binding to a phospholipid monolayer. To address the nonlinear spectra, we derive the theory of SFG response, when the interaction with bath is treated under cummulant expansion. We perform quantum mechanical calculations to determine the components of the Raman tensor and infrared transition dipole moments of the Amide I modes for three distinct structures hitherto reported for this molecule. We calculate the sum-frequency responses of Amide I modes of the fusion polypeptide for these different secondary structures and under different orientations in phospholipid membrane. The comparison of the modeled signals with that experimentally recorded allows us to address structural properties of this molecule under conditions which resemble those in vivo.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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Structural Properties of gp41 Fusion Peptide at a ... - ACS Publications

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