Membrane peptides :

1
Membrane peptides
2D-IR spectroscopy as a probe of structures and
dynamics
Yu-Shan Lin
Skinner Group
University of WisconsinMadison
2
Condensed phase vibrational spectroscopy Theory
and simulation
J. L. Skinner, B. M. Auer, Y.-S. Lin,
Vibrational line shapes and spectral diffusion
in liquid water, Adv. Chem. Phys. in press
Vibrational energy relaxation of the bend
fundamental of dilute water in liquid chloroform
and d-chloroform
1
Y.-S. Lin, S. G. Ramesh, J. M. Shorb, E. L.
Sibert III, J. L. Skinner, J. Phys. Chem. B 112,
390 (2008).
Water inertial reorientation Hydrogen bond
strength and the angular potential
2
D. E. Moilanen, E. E. Fenn, Y.-S. Lin, J. L.
Skinner, B. Bagchi, M. D. Fayer, Proc. Nat. Acad.
U.S.A 105, 5295 (2008).
Vibrational line shapes and spectral diffusion in
aqueous sodium bromide
3
Y.-S. Lin, B. M. Auer, J. L. Skinner, manuscript
in prepartion.
Membrane peptides 2D-IR Spectroscopy as a probe
of structures and dynamics
4
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009).
J. Manor, P. Mukherjee, Y.-S. Lin, H. Leonov, J.
L. Skinner, M. T. Zanni, I. T. Arkin, Structure
17, 247 (2009).
1
4
3
2
3
Condensed phase vibrational spectroscopy Theory
and simulation
Vibrational energy relaxation of the bend
fundamental of dilute water in liquid chloroform
and d-chloroform
1
Y.-S. Lin, S. G. Ramesh, J. M. Shorb, E. L.
Sibert III, J. L. Skinner, J. Phys. Chem. B 112,
390 (2008).
H2O in CHCl3
T1 8.50.5 ps
H2O in CDCl3
T1 28.51.0 ps
bend
(010)
Vibrational Energy Relaxation
(000)
4
Condensed phase vibrational spectroscopy Theory
and simulation
Vibrational energy relaxation of the bend
fundamental of dilute water in liquid chloroform
and d-chloroform
1
Water inertial reorientation Hydrogen bond
strength and the angular potential
2
D. E. Moilanen, E. E. Fenn, Y.-S. Lin, J. L.
Skinner, B. Bagchi, M. D. Fayer, Proc. Nat. Acad.
U.S.A 105, 5295 (2008).
5
Condensed phase vibrational spectroscopy Theory
and simulation
Vibrational energy relaxation of the bend
fundamental of dilute water in liquid chloroform
and d-chloroform
1
Water inertial reorientation Hydrogen bond
strength and the angular potential
2
Vibrational line shapes and spectral diffusion in
aqueous sodium bromide
3
Y.-S. Lin, B. M. Auer, J. L. Skinner, manuscript
in prepartion.
Fig. 1. Linear FT-IR spectra of the OD stretch of
HOD in pure water and aqueous NaBr solutions (H2O
background subtracted). n 8, 16, and 32 is the
number of water molecules per NaBr, and
correspond to approximately 6 M, 3 M, and 1.5 M
NaBr solutions, respectively.
S. Park, M. D. Fayer, Proc. Nat. Acad. U.S.A 104,
16731 (2007)
6
Condensed phase vibrational spectroscopy Theory
and simulation
Vibrational energy relaxation of the bend
fundamental of dilute water in liquid chloroform
and d-chloroform
1
Water inertial reorientation Hydrogen bond
strength and the angular potential
2
Vibrational line shapes and spectral diffusion in
aqueous sodium bromide
3
Membrane peptides 2D-IR spectroscopy as a probe
of structures and dynamics
4
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009).
J. Manor, P. Mukherjee, Y.-S. Lin, H. Leonov, J.
L. Skinner, M. T. Zanni, I. T. Arkin, Structure
17, 247 (2009).
N-methylacetamide
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
7
Membrane peptides 2D-IR spectroscopy as a probe
of structures and dynamics
Introduction
N-methylacetamide as a model molecule empirical
frequency map
Application 1 CD3-?
Application 2 M2
Application 3 hIAPP
N-methylacetamide
Application 4 Ovispirin
Aggregate of human islet amyloid polypeptides
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Antimicrobial peptide Ovispirin
8
Membrane peptides 2D-IR spectroscopy as a probe
of structures and dynamics
Introduction
N-methylacetamide as a model molecule empirical
frequency map
Application 1 CD3-?
Application 2 M2
Application 3 hIAPP
N-methylacetamide
Application 4 Ovispirin
Aggregate of human islet amyloid polypeptides
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Antimicrobial peptide Ovispirin
9
Peptides and proteins
FIGURE 1 The pentapeptide serylglycyltyrosylalanyl
leucine, or Ser-Gly-Tyr-Ala-Leu. Peptides are
named beginning with the amino-terminal residue,
which by convention is placed at the left. The
peptide bonds are shaded the R groups are in red.
10
Peptides and proteins
11
Understanding structures by spectroscopy
X-ray diffraction
Myoglobin
From Protein Structure and Function by Gregory A.
Petsko and Dagmar Ringe
NMR
Aprotinin, also known as bovine pancreatic
trypsin inhibitor, BPTI
From Protein Structure and Function by Gregory A.
Petsko and Dagmar Ringe
12
Understanding structures by spectroscopy
Significant obstacles for determination of the
native structures of membrane proteins using
X-ray and NMR
S. H. White, Protein Sci. 13, 1948 (2004)
13
Understanding structures by IR spectroscopy
A particularly strong vibrational transition is
called amide I (mostly carbonyl stretch of
peptide bond)
14
Amide I band
Amide I frequencies are sensitive to local
conformations and local environments
Amide I
Amide II
N-methylacetamide (NMA)
Amide I modes are all coupled to form an exciton
band
Band shapes and positions are sensitive to
secondary structure
However, the couplings make the spectroscopic
signature less local
J. L. R. Arrondo, A. Muga, J. Castresana, F. M.
Goni, Prog. Biophys. Mol. Biol. 59, 23 (1993)
M. F. DeCamp, L. DeFlores, J. M. McCracken, A.
Tokmakoff, K. Kwac, M. Cho, J. Phys. Chem. B 109,
11016 (2005)
15
Isotope labeling
Can obtain more specific and local information
with isotope labeling
13C produces 40 cm-1 red shift 13C18O produces
60 cm-1 red shift
49
13C18O
2D-IR spectroscopy greatly improves sensitivity
13C16O amide I
13C18O amide I of 49L
amide II
P. Mukherjee, A. T. Krummel, E. C. Fulmer, I.
Kass, I. T. Arkin, M. Y. Zanni, J. Chem. Phys.
120, 10215 (2004)
16
1D-IR vs 2D-IR
13C18O amide I of 49L
t1
t3
Tw
FT-IR signal µ2
(µ transition dipole strength)
2D-IR signal µ4
Able to tune the band position of the laser
pulses to study the transition of interest
P. Mukherjee, A. T. Krummel, E. C. Fulmer, I.
Kass, I. T. Arkin, M. Y. Zanni, J. Chem. Phys.
120, 10215 (2004)
17
2D-IR spectroscopy of isotope-edited peptides
31
Isotope labeling
2D-IR spectroscopy
P. Mukherjee, I. Kass, I. T. Arkin, M. T. Zanni,
JPCB 110, 24740 (2006)
18
2D-IR spectroscopy of isotope-edited peptides
34
Isotope labeling
2D-IR spectroscopy
P. Mukherjee, I. Kass, I. T. Arkin, M. T. Zanni,
JPCB 110, 24740 (2006)
19
2D-IR spectroscopy of isotope-edited peptides
31
34
11 single isotope labeling
38
39
41
43
44
45
46
2D-IR spectroscopy
49
53
Theoretical spectra and interpretation of the
results?
P. Mukherjee, I. Kass, I. T. Arkin, M. T. Zanni,
JPCB 110, 24740 (2006)
20
Membrane peptides 2D-IR spectroscopy as a probe
of structures and dynamics
Introduction
N-methylacetamide as a model molecule empirical
frequency map
Application 1 CD3-?
Application 2 M2
Application 3 hIAPP
N-methylacetamide
Application 4 Ovispirin
Aggregate of human islet amyloid polypeptides
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Antimicrobial peptide Ovispirin
21
NMAD/D2O as model system
D
D
D
D
D
D
D
N-methylacetamide
NMA
D
D2O solvent avoids water bend NMAD/D2O
Amide I for NMAD(g) 1717 cm-1
Amide I for NMAD/D2O 94 cm-1 red shift 28 cm-1
line width
22
Theoretical line shapes
Key ingredient
Frequency time-correlation function (FTCF)
Example FT-IR
frequency for an instantaneous configuration
trajectory

IR spectra
C(t)
frequency map
MD simulation
ab initio calculation
23
Theoretical line shapes
frequency map
Ab initio electric field frequency map from
NMAD/D2O clusters.
CHARMM
N-methylacetamide
TIP3P water
??lt?gt- ?g ?g1717 cm-1
J. R. Schmidt, S. A. Corcelli, J. L. Skinner, J.
Chem. Phys. 121, 8887 (2004)
24
Simplification of the 13-parameter fit
Original map (2004) had 13 parameters.
S13
CHARMM
N-methylacetamide
TIP3P water
Found that could get just as good map with two
parameters
Refit 200 NMAD-D2O clusters
?1017174670ECy-512ENy
S2
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
J. R. Schmidt, S. A. Corcelli, J. L. Skinner, J.
Chem. Phys. 121, 8887 (2004)
25
Issues regarding ab intio-based frequency maps
CHARMM
N-methylacetamide
TIP3P water
Level of theory and basis set
Cluster size
Compatibility of E-field calculations
Transferability to other solvents
Transferability to other force fields
26
Empirical amide I vibrational frequency map
Original map (2004) had 13 parameters.
Found that could get just as good map with two
parameters
Suggests empirical electric field map
?101717aECbEN
Empirical frequency map
Parameters a and b are optimized to minimize
J. R. Schmidt, S. A. Corcelli, J. L. Skinner, J.
Chem. Phys. 121, 8887 (2004)
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
27
Empirical amide I vibrational frequency map
Suggests empirical electric field map
?101717aECbEN
Empirical frequency map
Parameters a and b are optimized to minimize
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
28
Empirical amide I vibrational frequency map
Level of theory and basis set
Cluster size
Compatibility of E-field calculations
Transferability to other solvents
Transferability to other force fields
29
Membrane peptides 2D-IR spectroscopy as a probe
of structures and dynamics
Introduction
N-methylacetamide as a model molecule empirical
frequency map
Application 1 CD3-?
Application 2 M2
Application 3 hIAPP
N-methylacetamide
Application 4 Ovispirin
Aggregate of human islet amyloid polypeptides
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Antimicrobial peptide Ovispirin
30
TCR/CD3 complex
The T-cell receptor (TCR) complex with TCR-a and
TCR-ß chains, CD3 and ?-chain accessory molecules
and the co-receptor CD4 (CD stands for cluster of
differentiation)
31
IR Spectroscopy of CD3? Exp vs. Calc average
frequency
Isotope labeling

2D-IR spectroscopy
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
32
IR Spectroscopy of CD3? Exp vs. Calc average
frequency
Isotope labeling

2D-IR spectroscopy
MD simulation

Freq map
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
33
IR Spectroscopy of CD3? Exp vs. Calc average
frequency
Theoretical frequency shifts are in the correct
range
Theoretical frequency shifts show more variation
from residue to residue than the exp
loc two nearest-neighbor residues
nloc peptide atoms other than the two
nearest-neighbor residues
lipid all lipid atoms
water all water atoms
ion all counterions
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
34
Analysis of frequency shift
loc two nearest-neighbor residues
nloc peptide atoms other than the two
nearest-neighbor residues
lipid all lipid atoms
water all water atoms
ion all counterions
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
35
IR Spectroscopy of CD3? Exp vs. Calc 2D
diagonal line width
Exp
Isotope labeling

2D-IR spectroscopy
Calc
MD simulation

Freq map
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
36
Analysis of frequency time-correlation function
and linewidth
Fast component
from fluctuation of water hydration or/and
intrahelical hydrogen bonds
homogeneous linewidth Gh
Slow component
from peptide structural fluctuation
inhomogeneous linewidth Gi
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
G43-T47
37
Analysis of frequency time-correlation function
and linewidth
2D-IR diagonal widths primarily probe slow
frequency fluctuations (inhomogeneous broadening)
Fast component
from fluctuation of water hydration or/and
intrahelical hydrogen bonds
homogeneous linewidth Gh
Slow component
from peptide structural fluctuation
inhomogeneous linewidth Gi
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
G43-T47
38
Analysis of frequency time-correlation function
and linewidth
2D-IR diagonal widths primarily probe slow
frequency fluctuations (inhomogeneous broadening)
Exp
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
G43-T47
39
CD3? conclusions
Average frequencies probe average environment
2D-IR diagonal widths primarily probe slow
frequency fluctuations (inhomogeneous broadening)
Empirical frequency map lays a firm foundation
for study need more development
N-methylacetamide
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Aggregate of human islet amyloid polypeptides
Antimicrobial peptide Ovispirin
40
Membrane peptides 2D-IR spectroscopy as a probe
of structures and dynamics
Introduction
N-methylacetamide as a model molecule empirical
frequency map
Application 1 CD3-?
Application 2 M2
Application 3 hIAPP
N-methylacetamide
Application 4 Ovispirin
Aggregate of human islet amyloid polypeptides
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Antimicrobial peptide Ovispirin
41
M2 proton channel
M2 proton channel
Another four-helix trans-membrane peptide bundle
pH gating at neutral pH the proton channel is
closed, while at low pH the channel is open
Channel (and H transport) can be blocked by
amantadine, but most flu strains have mutated
(S31N) and are now resistant (do not bind
amantadine)
What is the gating mechanism?
E. De Clercq, Nature Rev. 5, 1015 (2006)
42
M2 proton channel
A. L. Stouffer, R. Acharya, D. Salom, A. S.
Levine, L. Di Costanzo, C. S. Soto, V. Tereshko,
V. Nanda, S. Stayrook, W. F. DeGrado, Nature 451,
596 (2008)
43
M2 proton channel
A. L. Stouffer, R. Acharya, D. Salom, A. S.
Levine, L. Di Costanzo, C. S. Soto, V. Tereshko,
V. Nanda, S. Stayrook, W. F. DeGrado, Nature 451,
596 (2008)
44
M2 proton channel
isotope labeling 2D-IR
For open channel, oscillation due to progression
around helix? Larger frequency fluctuations due
to solvent exposure?
Experiments on closed channel suggest helix
rotation. Confirmed by infrared dichroism
measurements.
J. Manor, P. Mukherjee, Y.-S. Lin, H. Leonov, J.
L. Skinner, M. T. Zanni, I. T. Arkin, Structure
17, 247 (2009)
45
Helix rotation is the gate?
Theoretical 2D-IR diagonal linewidths for low pH
open channel are in qualitative agreement with
experiment.
Channel Open (theoretical)
Theoretical oscillation supports idea of helix
rotation when channel closes
J. Manor, P. Mukherjee, Y.-S. Lin, H. Leonov, J.
L. Skinner, M. T. Zanni, I. T. Arkin, Structure
17, 247 (2009)
46
Helix rotation is the gate?
Theoretical 2D-IR diagonal linewidths for low pH
open channel are in qualitative agreement with
experiment
Theoretical oscillation supports idea of helix
rotation when channel closes
A. L. Stouffer, R. Acharya, D. Salom, A. S.
Levine, L. Di Costanzo, C. S. Soto, V. Tereshko,
V. Nanda, S. Stayrook, W. F. DeGrado, Nature 451,
596 (2008)
J. Manor, P. Mukherjee, Y.-S. Lin, H. Leonov, J.
L. Skinner, M. T. Zanni, I. T. Arkin, Structure
17, 247 (2009)
47
Helix rotation is the gate?
J. Manor, P. Mukherjee, Y.-S. Lin, H. Leonov, J.
L. Skinner, M. T. Zanni, I. T. Arkin, Structure
17, 247 (2009)
48
M2 conclusions
pH change induces helix rotation, which
closes/opens channel
Blocking residues are Asp-24 and Val-28, rather
than Trp-41
2D-IR is useful tool for complex bio problems
where X-ray and NMR are limited (such as membrane
proteins)
Theory can provide qualitative support more
work needed
N-methylacetamide
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Aggregate of human islet amyloid polypeptides
Antimicrobial peptide Ovispirin
49
Membrane peptides 2D-IR spectroscopy as a probe
of structures and dynamics
Introduction
N-methylacetamide as a model molecule empirical
frequency map
Application 1 CD3-?
Application 2 M2
Application 3 hIAPP
N-methylacetamide
Application 4 Ovispirin
Aggregate of human islet amyloid polypeptides
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Antimicrobial peptide Ovispirin
50
IAPP Islet amyloid polypeptide (amylin)
A 37-residue peptide hormone secreted by
pancreatic ß-cells
hIAPP KCNTA TCATQ RLANF LVHSS NNFGA ILSST NVGSN
TY
Islet amyloid is a characteristic pathological
finding in patients with type II diabetes
No amyloid fibril formation no diabetes
Do amyloid aggregates cause disease?
Or is disease caused by disruption of cell
membranes due to hIAPP as it aggregates?
J. W. M. Höppener, B. Ahrén, C. J. M. Lips, New
Engl. J. Med. 343, 411 (2000)
P. Westermark, U. Engström, K. H. Johnson, G. T.
Westermark, C. Betsholtz, Proc. Natl. Acad. Sci.
USA 87, 5036 (1990)
51
hIAPP aggregation
Many questions about aggregation mechanism
Does hIAPP misfold, then aggregate?
Does aggregation induce misfolding?
Does presence of membrane accelerate aggregation?
Does it change aggregation mechanism?
2D-IR as a probe of aggregation kinetics
Isotope labeling of selected residues
Automated 2D-IR spectra can be taken in minutes,
to probe how local environments change with
aggregation, with and without membranes
Proof of principle Compare 2D-IR spectra for
monomer and aggregate
52
hIAPP 1LysH 2CysH 3ASN 4Thr 5Ala 6Thr 7CysH 8Ala
9Thr 10Gln 11Arg 12Leu 13Ala 14Asn 15Phe 16Leu 17V
al 18HisB 19Ser 20Ser 21Asn 22Asn 23Phe 24Gly 25Al
a 26Ile 27Leu 28Ser 29Ser 30Thr 31Asn 32Val 33Gly
34Ser 35Asn 36Thr 37Tyr
Simulation of hIAPP monomer in water (with
Professor de Pablos group)
hIAPP
N
Thr4
Thr6
Ser19
HisB18
C
Ser20
Ser28
2D diagonal width
Ser28
Ser19
Ser28
Thr4
HisB18
Ser20
Thr6
53
hIAPP 1LysH 2CysH 3ASN 4Thr 5Ala 6Thr 7CysH 8Ala
9Thr 10Gln 11Arg 12Leu 13Ala 14Asn 15Phe 16Leu 17V
al 18HisB 19Ser 20Ser 21Asn 22Asn 23Phe 24Gly 25Al
a 26Ile 27Leu 28Ser 29Ser 30Thr 31Asn 32Val 33Gly
34Ser 35Asn 36Thr 37Tyr
Simulation of hIAPP in water (with Professor de
Pablos group)
hIAPP monomer
Ser19
N
Thr4
Thr6
Ser19
HisB18
C
Ser20
Ser28
Ser19
hIAPP KCNTA TCATQ RLANF LVHSS NNFGA ILSST NVGSN
TY
54
hIAPP conclusions
2D-IR should be sensitive enough to distinguish
among monomer and aggregate
Need experiment on monomer and aggregate
Need experiments on aggregation kinetics, with
and without membranes
N-methylacetamide
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Aggregate of human islet amyloid polypeptides
Antimicrobial peptide Ovispirin
55
Membrane peptides 2D-IR spectroscopy as a probe
of structures and dynamics
Introduction
N-methylacetamide as a model molecule empirical
frequency map
Application 1 CD3-?
Application 2 M2
Application 3 hIAPP
N-methylacetamide
Application 4 Ovispirin
Aggregate of human islet amyloid polypeptides
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Antimicrobial peptide Ovispirin
56
Ovispirin
An 18-residue antimicrobial peptide
KNLRRIIRKIIHIIKKYG
Antimicrobial peptides are often ?-helices, with
hydrophilic and hydrophobic faces
They are often highly charged Ovispirin is 7,
due to the arginines and lysines
Such peptides are attracted to negatively charged
bacterial membranes, and destroy them
Mechanisms of antimicrobial action
barrel-stave model
toroidal model
carpet model
K. A. Brogden, Nature Rev. 3, 238 (2005)
57
Ovispirin
How does Ovispirin sit in/on membrane?
Solid-state NMR can provide information about
orientation
How deep? Can 2D-IR on isotope-labeled peptides
provide the information about depth?
Experiments in progress (Professor Zannis group)
Simulations in progress
Theory Exp to identify most likely position
Investigate mechanism of antimicrobial action?
S. Yamaguchi, D. Huster, A. Waring, R. I. Lehrer,
W. Kearney, B. F. Tack, M. Hong, Biophys. J. 81,
2203 (2001)
58
Membrane peptides 2D-IR spectroscopy as a probe
of structures and dynamics
Introduction
N-methylacetamide as a model molecule empirical
frequency map
Application 1 CD3-?
Application 2 M2
Application 3 hIAPP
N-methylacetamide
Application 4 Ovispirin
Aggregate of human islet amyloid polypeptides
CD3-? transmembrane peptide bundle in T-cell
receptor complex
M2 proton channel in influenza A virus
Antimicrobial peptide Ovispirin
59
Acknowledgements
Current group members
Professor Jim Skinner
Professor Mino Yang
Dr. Piotr Pieniazek
Fu Li
Lu Wang
Craig Tainter
Pallavika Ramaswamy
Professor J. R. Schmidt
Justin Shorb
Professor Juan de Pablo
Professor Martin Zanni
Professor Shy Arkin
A. Santosh Reddy
Dr. Prabuddha Mukherjee
Hadas Leonov
David Strasfeld
Ann Woys
National Science Foundation
National Institutes of Health
60
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61
Condensed phase vibrational spectroscopy Theory
and simulation
Vibrational energy relaxation of the bend
fundamental of dilute water in liquid chloroform
and d-chloroform
1
Y.-S. Lin, S. G. Ramesh, J. M. Shorb, E. L.
Sibert III, J. L. Skinner, J. Phys. Chem. B 112,
390 (2008).
H2O in CHCl3
T1 8.50.5 ps
H2O in CDCl3
T1 28.51.0 ps
Vibrational Energy Relaxation
62
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63
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64
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65
Condensed phase vibrational spectroscopy Theory
and simulation
Water inertial reorientation Hydrogen bond
strength and the angular potential
2
D. E. Moilanen, E. E. Fenn, Y.-S. Lin, J. L.
Skinner, B. Bagchi, M. D. Fayer, Proc. Nat. Acad.
U.S.A 105, 5295 (2008).
66
Analysis of frequency time-correlation function
and linewidth
2D-IR diagonal widths primarily probe slow
frequency fluctuations (inhomogeneous broadening)
Fast component
from fluctuation of water hydration or/and
intrahelical hydrogen bonds
homogeneous linewidth Gh
Slow component
43Gly
from peptide structural fluctuation
47Thr
inhomogeneous linewidth Gi
Y.-S. Lin, J. M. Shorb, P. Mukherjee, M. T.
Zanni, J. L. Skinner, J. Phys. Chem. B 113, 592
(2009)
G43-T47
67
Analysis
G43-T47
68
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69
Amide I band in peptides
FIGURE 3-14 The pentapeptide serylglycyltyrosylala
nylleucine, or Ser-Gly-Tyr-Ala-Leu. Peptides are
named begginning with the amino-terminal residue,
which by convention is placed at the left. The
peptide bonds are shaded the R groups are in red.
J. L. R. Arrondo, A. Muga, J. Castresana, F. M.
Goni, Prog. Biophys. Mol. Biol. 59, 23 (1993)
70
Amide I frequencies are sensitive to secondary
structures
J. L. R. Arrondo, A. Muga, J. Castresana, F. M.
Goni, Prog. Biophys. Mol. Biol. 59, 23 (1993)
M. Cho, Nature 444, 431 (2006)
71
IR spectroscopy of isotope-edited CD3?
amide I
ester stretch of membrane headgroups
49
13C18O
13C16O amide I 49L
amide II
13C18O amide I 49L
P. Mukherjee, A. T. Krummel, E. C. Fulmer, I.
Kass, I. T. Arkin, M. Y. Zanni, J. Chem. Phys.
120, 10215 (2004)
72
Time-scale in NMR Spectroscopy
Wavelength of radiofrequency wave 1 m
Frequency of radiofrequency wave c/? 3x108 s-1
One cycle of radiofrequency wave (3x108)-1
3.3x10-9 s 3.3 ns
The pulse duration limits the time resolution of
the spectroscopy
The time resolution of NMR spectroscopy 10 ns
73
Time-scale in IR Spectroscopy
The time resolution of IR spectroscopy 50 fs
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M2 proton channel
Adamantane (tricyclo3.3.1.13,7decane)
E. De Clercq, Nature Rev. 5, 1015 (2006)
77
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HE stain
HE stain, HE stain or hematoxylin and eosin
stain, is a popular staining method in histology.
It is the most widely used stain in medical
diagnosis for example when a pathologist looks
at a biopsy of a suspected cancer, the
histological section is likely to be stained with
HE and termed HE section, HE section, or HE
section. The staining method involves
application of the basic dye hematoxylin, which
colors basophilic structures with blue-purple
hue, and alcohol-based acidic eosin Y, which
colors eosinophilic structures bright pink. The
basophilic structures are usually the ones
containing nucleic acids, such as the ribosomes
and the chromatin-rich cell nucleus, and the
cytoplasmatic regions rich in RNA. The
eosinophilic structures are generally composed of
intracellular or extracellular protein. The Lewy
bodies and Mallory bodies are examples of
eosinophilic structures. Most of the cytoplasm is
eosinophilic. Red blood cells are stained
intensely red. The structures do not have to be
acidic or basic to be called basophilic and
eosinophilic. The terminology is based on the
affinity to the dyes. Other colors, e.g. yellow
and brown, can be present in the sample they are
caused by intrinsic pigments, e.g. melanin. Some
structures do not stain well. Basal laminae need
to be stained by PAS stain or some silver stains,
if they have to be well visible. Reticular fibers
also require silver stain. Hydrophobic structures
also tend to remain clear these are usually rich
in fats, eg. adipocytes, myelin around neuron
axons, and Golgi apparatus membranes.
79
Model membranes need to be negatively charged
use lipid mixtures

• POPCPOPG 31

POPC
Simulations with lipid mixtures are hard to
equilibrate! (DePablo)
80
DMPC 1,2-dimyristol-sn-glycero-phosphatidylcholin
e
81
Local amide I mode in peptides
Method1 fully electrostatic
Method2 f-? map electrostatic
from a f-? map
Implement 1-4 exclusion principle to avoid
artifacts from atoms too close
Tyr-33
Asp-36
Thr-47
T. La Cour Jansen, A. G. Dijkstra, T. M. Watson,
J. D. Hirst, J. Knoester, JCP 125, 044312 (2006)
82
Calculation of IR spectra
Frequency time-correlation function
FT-IR
2D-IR
T10, T21 vibrational relaxation times
83
N. Demirdoven, C. M. Cheatum, H. S. Chung, M.
Khalil, J. Knoester, A. Tokmakoff, J. Am. Chem.
Soc. 126, 7981 (2004)
84
hIAPP 1LysH 2Cys2 3ASN 4Thr 5Ala 6Thr 7Cys2 8Ala
9Thr 10Gln 11Arg 12Leu 13Ala 14Asn 15Phe 16Leu 17V
al 18HisB 19Ser 20Ser 21Asn 22Asn 23Phe 24Gly 25Al
a 26Ile 27Leu 28Ser 29Ser 30Thr 31Asn 32Val 33Gly
34Ser 35Asn 36Thr 37Tyr
Simulation of hIAPP in water (with Professor de
Pablos group)
monomer
aggregate
hIAPP KCNTA TCATQ RLANF LVHSS NNFGA I LSST
NVGSN TY-NH2
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hIAPP 1LysH 2Cys2 3ASN 4Thr 5Ala 6Thr 7Cys2 8Ala
9Thr 10Gln 11Arg 12Leu 13Ala 14Asn 15Phe 16Leu 17V
al 18HisB 19Ser 20Ser 21Asn 22Asn 23Phe 24Gly 25Al
a 26Ile 27Leu 28Ser 29Ser 30Thr 31Asn 32Val 33Gly
34Ser 35Asn 36Thr 37Tyr
Simulation of hIAPP monomer in water (with
Professor de Pablos group)
folded
misfolded
random coil
ß-hairpin
aggregate precursor
hIAPP KCNTA TCATQ RLANF LVHSS NNFGA I LSST
NVGSN TY-NH2