Developing 2D IR Spectroscopy as a
Quantitative Probe for Protein Structure
Ann Marie Woys
University of Wisconsin – Madison
June 25, 2010
Membrane polypeptides & proteins are an important class of
biomolecules AND extremely difficult to study.
3 examples of systems in which both structure and dynamics are important.
Antibiotics
– Various mechanisms, steps include
• Binding
• Pore formation
• Lysis
L. Gottler, A. Ramamoorthy, Biochim. Biophys. Acta, 1788, 1680–1686, (2009).
KcsA Channel Selectivity Filter
– Selective for K+ over Na+, due to
electrostatics, structure and
dynamics of selectivity filter.
H+
H+
Closed
H+
Closed +
Protons
H+
H+
Open
Conductive
H+
H+
Open +
Inactivated
Amyloids
– The fibers of many amyloid peptides
are catalyzed by membranes
– Intermediates are toxic
S. Shim, PNAS, 106, 6614–6619 (2009).
However, extremely difficult to study with standard structural techniques.
Trends in EPR linewidths provide information on
secondary structure and molecular assembly.
Surface adsorbed
helix
Our goal:
See if we can use
IR spectroscopy
to get similar
information but
without
mutations.
polar
nonpolar
sequence
position
Water soluble
protein
polar
nonpolar
sequence
position
G. Fanucci, D. Cafiso Curr. Opin. Struct. Biol., 16, 644–653 (2006).
M. Apostolidou, S. Jayasinghe, R. Langen, J. Biol. Chem., 283, 17205–17210 (2008).
W. Hubbell, A. Gross, R. Langen, M. Lietzow. Curr. Opin. Struct. Biol. 8, 649-656 (1998).
Transmembrane
water-filled pore
polar
nonpolar
sequence
position
Can infrared spectroscopy do this?
• Amide I Band
– Has different line
widths for
membrane versus
soluble peptides
• Implies
environmental
sensitivity
– Can isotope label to
resolve individual
residues
•
13C18O
Membrane peptides span a wide
range of environments.
• Electrostatics
• Does it alter lifetime of amide I?
• Hydrogen bonding
• Or vibrational dynamics?
• Large concentration gradients • How do we quantify this?
S. White and W. Wimley, Annu. Rev. Biophys. Biomol. Struct. 28, 319 (1999).
2D IR spectroscopy measures lifetime & vibrational
dynamics.
Vibrational dynamics
Homogeneous
Inhomogeneous
Ovispirin – Hydrophobic vs. Hydrophilic Residue
KNLRR IIRKI IHIIK KYG
Ovispirin
Homogeneous & Inhomogeneous Linewidth
• Homogeneous
linewidth
5-7 cm-1
– No institutive
oscillations.
• e.g. no clear
correlation to peptide
structure or membrane
environment
• Inhomogeneous
linewidth
8-24 cm-1
– Avg. of about 13 cm-1
– Most importantly, it is
periodic.
Ovispirin – 2D IR Diagonal Linewidths
KNLRR IIRKI IHIIK KYG
• Results
– Period is 3.6 residues (α-helix)
– Similar to extended wheel diagram
prediction
– Hydrophilic residues have largest
linewidth.
– Hydrophobic have smallest.
– Clear intuitive correlation between
experiment and structure.
• Notice: Trend is lower in the center.
Maybe tilted in bilayer and
kinked?
Ovispirin – Filling in Structural Details with MD
Simulations
Collaboration with Juan
dePablo & Jim Skinner
•
•
Tilted in bilayer: deeper Nterminus
Kinked at residue 12
Potential Mean Force
Backbone depth
Ovispirin – Filling in Structural Details with MD
Simulations
Collaboration with Juan
dePablo & Jim Skinner
•
•
Tilted in bilayer: deeper Nterminus
Kinked at residue 12
Potential Mean Force
Backbone depth
Ovispirin – Calculating 2D Spectra Using MD Predicted
Structure
•
•
•
•
•
•
•
Simulations also predict 3.6
residue oscillations
Trend correlates to peptide tilt
in bilayer.
Similar average value and
range
Same periodic trend near Nterminus
Maybe MD tilt is not correct.
Comparison not as good at Cterminus beginning at kink
(~res. 12)
And the kink may explain
trend in experimental data.
Remember: EPR Trends
Surface adsorbed
helix
polar
nonpolar
sequence
position
Water soluble
protein
Transmembrane
water-filled pore
polar
nonpolar
sequence
position
polar
nonpolar
sequence
position
W. Hubbell, A. Gross, R. Langen, M. Lietzow. Curr. Opin. Struct. Biol. 8, 649-656 (1998).
Summary - Produces Picture Like from EPR Paper
Ovispirin
CD3ζ
M2
2D IR Spectroscopy for Membrane Protein/Peptide
Structure
• Inherent advantages of isotope
labeling
– Native probe: can put anywhere
– Spectra calculated from
molecular dynamics simulation
– IR probes a local environment
• Hydration, backbone
fluctuations, electrostatic
environment
– Use to study dynamics/kinetics
• But, some drawbacks with 13C18O
–
–
–
–
Overlaps with some side chains
Limited to proteins <120 residues
Requires semi-synthesis of proteins
In the future, for larger proteins, we
will label with a metal carbonyl tag
Acknowledgments
Martin Zanni
–
–
–
–
–
–
–
–
Chris Middleton
Sean Moran
Emily Blanco
Sudipta Mukherjee
Lauren Buchanan
Ha Dong
Jenny Laaser
David Skoff
• Jim Skinner
– Yu-Shan Lin
• Juan dePablo
– A. Santosh Reddy
Is it possible to get the presented results from FTIR
spectroscopy?
FTIR
|μ|2
2DIR
|μ|4
• Maybe (we haven’t been able to - background).
• 2D IR intensity
– Minimizes broad background peaks (e.g. water)
Convert to 2D IR Spectra Using Skinner Method
Measure electric field within 20 Å
radius for C and N atoms
Convert to frequencies
Use frequencies over 2 ns 20 times
within 200 ns trajectory to get the
correlation function
Calculate response function
Calculate 2D IR spectrum
Ovispirin – Sigma Decomposition
• σ2 - distribution of frequency
fluctuations
– Does not include dynamics (line
narrowing)
• Peptide, lipid water - all
periodic
• But cross terms are the most
important contribution, also
have periodic trend
Clearly, vibrational dynamics are
very different on one side of helix
than the other, due to the
environment, but cannot assign
to a specific contribution.
Everything working in tandem.
Nonetheless, result is still intuitive.
2D IR inhomogeneous linewidth
scales with electrostatic disorder.
Increased Lipid Concentration Does Not Effect
Experimental Linewidth
Effect of Mutation on Peptide Depth & Linewidth