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