Developing 2D IR Spectroscopy as a Quantitative Probe for Protein Structure Ann Marie Woys...
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Transcript of Developing 2D IR Spectroscopy as a Quantitative Probe for Protein Structure Ann Marie Woys...
Developing 2D IR Spectroscopy as a Quantitative Probe for Protein Structure
Ann Marie WoysUniversity of Wisconsin – MadisonJune 25, 2010
Membrane polypeptides & proteins are an important class of biomolecules AND extremely difficult to study.
Antibiotics– Various mechanisms, steps
include • Binding• Pore formation• Lysis
KcsA Channel Selectivity Filter– Selective for K+ over Na+, due
to electrostatics, structure and dynamics of selectivity filter.
Amyloids– The fibers of many amyloid
peptides are catalyzed by membranes
– Intermediates are toxicHowever, extremely difficult to study with standard structural techniques.
3 examples of systems in which both structure and dynamics are important.
Closed Closed + Protons
Open Conductive
Open + Inactivated
H+ H+ H+ H+ H+ H+
H+
L. Gottler, A. Ramamoorthy, Biochim. Biophys. Acta, 1788, 1680–1686, (2009).
S. Shim, PNAS, 106, 6614–6619 (2009).
Trends in EPR linewidths provide information on secondary structure and molecular assembly.
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).
Water soluble protein
sequence position
polarnonpola
r
Transmembrane water-filled
pore
sequence position
polarnonpola
r
Surface adsorbed helix
sequence position
polarnonpola
r
Our goal: See if we can use IR spectroscopy to get similar information but without mutations.
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• Hydrogen bonding• Large concentration
gradients
• Does it alter lifetime of amide I?
• Or vibrational dynamics?• 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
Backbone depthPotential Mean Force
Collaboration with Juan dePablo & Jim Skinner
• Tilted in bilayer: deeper N-terminus
• Kinked at residue 12
Ovispirin – Filling in Structural Details with MD Simulations
Backbone depthPotential Mean Force
Collaboration with Juan dePablo & Jim Skinner
• Tilted in bilayer: deeper N-terminus
• Kinked at residue 12
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 N-terminus
• Maybe MD tilt is not correct.• Comparison not as good at C-
terminus beginning at kink (~res. 12)
• And the kink may explain trend in experimental data.
Remember: EPR Trends
Water soluble protein
sequence position
polarnonpola
r
Transmembrane water-filled
pore
sequence position
polarnonpola
r
Surface adsorbed helix
sequence position
polarnonpola
r
W. Hubbell, A. Gross, R. Langen, M. Lietzow. Curr. Opin. Struct. Biol. 8, 649-656 (1998).
Ovispirin
Summary - Produces Picture Like from EPR Paper
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?
• Maybe (we haven’t been able to - background).• 2D IR intensity
– Minimizes broad background peaks (e.g. water)
FTIR|μ|2
2DIR|μ|4
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
Nonetheless, result is still intuitive. 2D IR inhomogeneous linewidth scales with electrostatic disorder.
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.
Increased Lipid Concentration Does Not Effect Experimental Linewidth
Effect of Mutation on Peptide Depth & Linewidth