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Developing 2D IR Spectroscopy as a Quantitative Probe for Protein Structure Ann Marie Woys University of Wisconsin – Madison June 25, 2010.

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Presentation on theme: "Developing 2D IR Spectroscopy as a Quantitative Probe for Protein Structure Ann Marie Woys University of Wisconsin – Madison June 25, 2010."— Presentation transcript:

1 Developing 2D IR Spectroscopy as a Quantitative Probe for Protein Structure Ann Marie Woys University of Wisconsin – Madison June 25, 2010

2 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 toxic However, 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+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).

3 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, (1998). Water soluble protein sequence position polar nonpolar Transmembrane water-filled pore sequence position polar nonpolar Surface adsorbed helix sequence position polar nonpolar Our goal: See if we can use IR spectroscopy to get similar information but without mutations.

4 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 13 C 18 O

5 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).

6 2D IR spectroscopy measures lifetime & vibrational dynamics. Vibrational dynamics HomogeneousInhomogeneous

7 Ovispirin – Hydrophobic vs. Hydrophilic Residue KNLRR IIRKI IHIIK KYG

8 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.

9 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?

10 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

11 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

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.

13 Remember: EPR Trends Water soluble protein sequence position polar nonpolar Transmembrane water-filled pore sequence position polar nonpolar Surface adsorbed helix sequence position polar nonpolar W. Hubbell, A. Gross, R. Langen, M. Lietzow. Curr. Opin. Struct. Biol. 8, (1998).

14 Ovispirin Summary - Produces Picture Like from EPR Paper CD3ζM2

15 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 13 C 18 O – 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

16 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

17 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

18 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

19 Ovispirin – Sigma Decomposition σ 2 - 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.

20 Increased Lipid Concentration Does Not Effect Experimental Linewidth

21 Effect of Mutation on Peptide Depth & Linewidth


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