2D IR Spectroscopy Reveals the Role of Water in the Binding of
Channel-Blocking Drugs to the Influenza M2 Channel
Ayanjeet Ghosh*, Jun Wang, Yurii S. Moroz, Ivan V. Korendovych, Martin Zanni, William F.
DeGrado, Feng Gai*, and Robin M. Hochstrasser#
CD spectra and DLS measurements of M2: To check for proper folding and tetramerization,
unlabeled R45A M2 samples constituted as described above were subjected to circular dichroism
(CD) and dynamic light scattering (DLS) measurements (Figures S1 and S2). As shown in
Figure S1, the CD spectrum indicates that the R45A M2 sequence forms well-defined helices as
expected from previous studies.[1] The DLS experiment shows a narrow distribution of micelle
sizes (Figure S2), with the major population (~93%) corresponding to a radius of 24 nm, which
is close to values previously reported using vesicles.[2] This suggests well-formed tetramers, as
the micelle size for monomers is expected to be smaller than that for a tetrameric channel.
Moreover, the pH and drug dependence seen in the 2D IR spectra also indicate a well-formed
tetramer, as a monomer is not expected to show any significant pH dependence in its spectra.[3]
Figure S1. CD spectrum of R45A M2, which was collected on an Aviv 62A DS
spectropolarimeter (Aviv Associates, NJ) with a 1 mm sample holder. The peptide concentration
was ~50-80 μM in phosphate buffer (pH* 7, D2O).
i
Figure S2. Micelle size distributions and their corresponding percent populations for R45A M2
at pH 7, which were determined by measuring the autocorrelation of scattering intensity at 782.4
nm using a DynaPro99 instrument (Wyatt, formerly Protein Solutions) with a 3-window black
quartz cuvette (Hellma, Kent, UK). The sample concentration was between 50-80 μM.
Fitting Diagonal Traces of 2D IR Spectra: The diagonal traces were obtained from 2D IR
spectra of the labeled Ala30 and Gly34 modes at pH 5 at waiting time T = 0. For all cases the
signal along a line parallel to the diagonal line was selected which runs through the positive peak
maxima. A linear baseline was subtracted from all traces. The traces for both Gly34 and Ala30
were fit to two Gaussian functions given as:
y  A1e
 (  1 )2
212
 A2e
 (  2 )2
2 22
(1)
where A1 and A2 are amplitude factors that were allowed to assume any integral value between 1
and 3. All the data fit well to a model where A1:A2 = 1:3. The parameters of the fits are shown in
Table S1.
ii
Figure S3. Experimental diagonal traces (black circles) from 2D IR spectra of Ala30 and Gly34
at pH 5 under different drug binding conditions. The fits are shown as red solid lines. A, B, C:
Diagonal traces for Gly34 for drug free, rimantadine (Rim) bound and spiran amine (SA) bound
channels, respectively. D, E, F: Diagonal traces for Ala30 for drug free, rimantadine (Rim)
bound and spiran amine (SA) bound channels, respectively.
Table S1. Fitted parameters for 2D diagonal traces shown in Figure S3. All parameters have
units of wavenumbers.
Ala30
Gly34
ω1
σ1
ω2
σ2
ω1
σ1
ω2
σ2
Drug Free
1571
5
1584.5
6.1
1591
5
1574
6.8
Rim bound
1570
5
1584
7.5
1590.5
5
1574.3
7.4
SA bound
1567
5
1582
6.8
1590.2
6.7
1574
7.3
Fitting 2D IR Intensities and Peak Shifts for Estimation of T1 and τc: The 2D IR signal
decay was evaluated by recording the relaxation of the 2D peak intensities with the waiting time,
T. The peak shifts were evaluated at each waiting time by windowing out the amide transition
and subsequently Fourier transforming the signal to the mixed time-frequency domain S(τ,ωt,T)
and then integrating over the ωt domain, thus yielding a signal S(τ,T). The position of the
maxima of this signal was recorded, and the procedure was repeated for all waiting times. Both
the 2D intensity relaxation data and the peak shift data were normalized to the value of the
corresponding metric at T = 0. It is well known that the 2D signal intensity shows exponential
decay due to T1 relaxation.[4] It has been also been shown that the evolution of the peak shifts
iii
mirror the frequency correlation function, which is an exponential function for Gaussian
frequency fluctuations.[4] Both the 2D peak intensities and the peak shifts were fit to an
T
exponential decay of the form: y  a  e b  c to obtain the vibrational lifetime T1 and the
frequency correlation time τC, respectively. The fits result in the offset c being zero for all cases,
which is expected from reports in the literature.[5] The fitted values of T1 and τC and their error
bars for the different experimental cases are shown in Table S2 and S3.
Table S2. T1 values from fits to the 2D peak intensities shown in Figure 4.
Condition
Drug Free, pH 7
T1 (ps)
Gly34
1.1 ± 0.3
T1 (ps)
Ala30
1.13 ± 0.09
Rim bound, pH 7
1.18 ± 0.1
1.15 ± 0.2
SA bound, pH 7
Drug Free, pH 5, low frequency
Rim bound, pH 5, low frequency
1.16 ± 0.2
1.0 ± 0.25
0.97 ± 0.1
1.09 ± 0.2
-
SA bound, pH 5, low frequency
0.92 ± 0.7
-
Drug Free, pH 5, high frequency
1.24 ± 0.7
-
Rim bound, pH 5, high frequency
SA bound, pH 5, high frequency
1.2 ± 0.2
1.2 ± 0.4
-
Table S3. τC values from fits to the 2D peak shifts shown in Figure 6.
Condition
c (ps)
c (ps)
Drug Free, pH 7
Gly34
5±1
Ala30
7±4
Rim bound, pH 7
2.7 ± 0.6
2.8 ± 1
SA bound, pH 7
2.5 ± 0.7
3.7 ± 0.8
Simulations of 2D IR spectra: To test whether the peak shifts evaluated from experimental 2D
IR spectra can reproduce the correlation decay with high fidelity, numerical simulations of 2D IR
spectra of two overlapping transitions were performed. The third-order responses used for these
calculations have been derived and detailed elsewhere and hence are not repeated. A vibrational
frequency correlation function (FCF) of the form:
C (t )  12 e
 t
h
  22e
iv
 t
c
 2
was used, where 12 e
and the term  22 e
t
t
C
h
is the homogeneous component, σ is the inhomogeneous component,
is the Kubo function that causes the FCF to decay. For one of the
transitions, Δ2 was set to zero, while for the other Δ2 = 25 cm-1 and τ = 1.5 ps. Δ1 and σ were set
to 5 cm-1 and  h to 50 fs for both modes. The transition frequencies are separated by 15 cm-1. The
transition of interest was windowed out following the same procedure used for experimental
data. The resulting peakshift relaxation yields a timescale of 1.3 ps, which reproduces the actual
decay time of 1.5 ps reasonably well.
Figure S4. Peakshift decay from the simulated 2D IR spectra of two overlapping transitions.
Linear IR spectra of the isotope label: The linear IR spectra at pH 5 in isotope labeled region
shows the same asymmetry observed in the 2D diagonal traces. Since 2D IR offers better
signal:background ratio, the 2D diagonal traces were used to evaluate the relative populations of
these substates. The following spectrum shows the asymmetry of the Ala30 band at pH5 in the
drug free channel. A linear baseline was subtracted from the spectrum. The FTIR spectra of the
other pH 5 samples also show the asymmetry observed in 2D IR measurements.
v
Figure S5. FTIR spectrum of the isotope labeled A30 amide at pH 5 in the drug free channel. A
linear baseline was subtracted from the spectrum.
References
[1]
D. Salom, B. R. Hill, J. D. Lear, W. F. DeGrado, Biochemistry-Us 2000, 39, 14160.
[2]
T. Wang, S. D. Cady, M. Hong, Biophys J 2012, 102, 787.
[3]
M. Reppert, A. Tokmakoff, J Chem Phys 2013, 138.
[4]
P. Hamm, M. T. Zanni, Concepts and methods of 2d infrared spectroscopy, Cambridge
University Pres, Cambridge ; New York, 2011.
[5]
D. G. Kuroda, J. D. Bauman, J. R. Challa, D. Patel, T. Troxler, K. Das, E. Arnold, R. M.
Hochstrasser, Nature Chemistry 2013, 5, 174-181; bJ. T. King, M. R. Ross, K. J.
Kubarych, J Phys Chem B 2012, 116, 3754.
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