Physiologically-relevant crowding effects on a protein-peptide interaction. Sam Stadmiller Pielak Lab UNC Chapel Hill April 10, 2018

Transcription

1 Physiologically-relevant crowding effects on a protein-peptide interaction Sam Stadmiller Pielak Lab UNC Chapel Hill April 10, 2018

2 From fundamentals to applications Biophysics of cell signaling Computation Implications Yu et al. elife. (2016) Biotherapeutic design and development Energy calculations Experimental validation & refinement

3 Cells are crowded and dynamic Most experiments Proteins native environment Buffer < 10 g/l ~ 300 g/l macromolecules Yu et al. elife. (2016)

4 Protein-protein interactions are essential to life A. Garrow, Human Interactome, 2006,

5 SH3 interactions are important for cell signaling Mitogen activated protein kinase (MAPK) signaling Activated RTK Exterior Cytosol Activated Ras G T P SOS N-SH3 C-SH3 SH2 P MAPK SIGNALING Adapted from: Lodish, Molecular Cell Biology

6 Protein and peptide of interest N-terminal SH3 domain from adaptor protein SOS peptide Sequence PDB 2A kda pi kda pi

7 Predicted bound structure Kurcinski, M. et al. Nuc. Acids Res. (2015)

8 19 F labeling of SH3 Trp 36 5F-Trp 36 5-fluoroindole + IPTG 19 F NMR advantages 100% abundant Sensitivity (83% proton) Rare in biology Easily incorporated into proteins

9 Peptide binding affects 19 F chemical environment k on k off + k ex = k on [peptide] + k off Bound Free

10 NMR lineshapes are information-rich Free Bound ω B p B ω A p A R 2B R 2A with Chris Waudby Lineshape = solution to Bloch equations

11 NMR lineshapes are information-rich [peptide] Free Bound ω B p B R 2B Lineshape = solution to Bloch- McConnell equations ω A p A R 2A with Chris Waudby K D, k ex, [SH3]=constant, [peptide]

12 Protein association and free energy diagrams TS G + Reaction coordinate

13 Protein association and free energy diagrams ΔG D = ΔG D - ΔGA ΔG D = RT ln K D ΔG A TS ΔG k h A/D= RTln k B T G Free ΔG D ΔG D Bound Reaction coordinate

14 SH3-peptide interaction in buffer ΔG D = ΔG D - ΔGA k on = 1.4 x 10 8 M -1 s -1 ΔG D = RT ln K D 6.8 ± 0.1 TS ΔG k h A/D= RTln k B T G (kcal/mol) Free 5.6 ± ± 0.1 k off = 2.1 x 10 4 s -1 K D = 150 µm Bound Reaction coordinate

15 Investigating macromolecular crowding effects Lysozyme Ovalbumin BSA PDB 5B1F PDB 1OVA PDB 4F5S MW (kda) pi Charge at ph

16 Electrostatic crowder-sh3 interactions slightly perturb binding affinity ΔΔG D = ΔG D,crowder ΔG D,buffer

17 Electrostatic crowder-sh3 interactions slightly perturb binding affinity ΔΔG D = ΔG D,crowder ΔG D,buffer Stabilizes complex Destabilizes complex

18 Crowder charge dictates kinetic effects ΔΔG A = ΔG A, crowder ΔG A,buffer

19 Crowder charge dictates kinetic effects ΔΔG A = ΔG A, crowder ΔG A,buffer Increases barrier to association Decreases barrier to association Only positively charged lysozyme affects the association rate constant

20 Crowder charge dictates kinetic effects ΔΔG D = ΔG D, crowder ΔG D,buffer

21 Crowder charge dictates kinetic effects ΔΔG D = ΔG D, crowder ΔG D,buffer Increases barrier to dissociation Decreases barrier to dissociation Only high concentrations of negatively charged BSA affects the dissociation rate constant

22 Future Crowded Conditions Lysozyme BSA + Ovalbumin PDB 1GB1 Cell lysates Investigate concentration and size dependence (if any)

23 From fundamentals to applications Biophysics of cell signaling Computation Implications Yu et al. elife. (2016) Biotherapeutic design and development Energy calculations Experimental validation & refinement

24 Acknowledgments Pielak Lab NMR Greg Young Lineshape Analysis Chris Waudby, UCL