Lipid Monolayer Response to Lateral Stress the Role of Lung Surfactant Protein B Luka Pocivavsek1, Shelli Frey1, Josh Kurutz2, Kapil Krishan3, Alan Waring4, Ka Yee Lee1 1Department of Chemistry, James Franck Institute, Institute for Biophysical Dynamics, and MRSEC, University of Chicago 2Department of Biochemistry NMR Facility, University of Chicago 3Department of Physics and Astronomy, UC Irvine 4Department of Pediatrics, Harbor/UCLA Medical Center

Outline introduction to lung surfactant and our model system description of how lung surfactant peptides can impact the response of the lipid monolayer to lateral stress and the questions we pose in elucidating the role of these peptides and especially the N-terminus FM and AFM data is presented that show the effect of lung surfactant peptides on 1. monolayer phase behavior, 2. monolayer fluidity/jamming, 3. monolayer collapse modes solved NMR structures for two of the peptides, SPB 9-25 and 11-25, are presented conclusions

Lung Surfactant and SP-B LS is composed of lipids and proteins - we study a model system composed of a binary lipid mixture and SPB truncation peptides: DPPC POPG SP-B 1-25: FPIPLPYCWLCRALIKRIQAMIPKG WT SP-B 9-25: WLCRALIKRIQAMIPKG N-terminus SP-B 11-25: CRALIKRIQAMIPKG tryptophane SP-B 1-25 Nflex: FGIGLPYCWLCRALIKRIQAMIPKG N-flexibility SP-B 1-25 neut: FPIPLPYCWLCAALIAAIQAMIPAG cationic charge dimensions DPPC:POPG 7:3 10% wt/wt peptide Charge has already been proven to play a major role in SPB function, however the role of the 11 N-terminal amino acids has not been elucidated. Thus we designed three mutant peptides to explore the role of the N-terminus.

How does SP-B modulate the monolayer response Questions Addressed How does SP-B modulate the monolayer response to lateral stress and the mode of stress relaxation? how do the different peptides affect the phase behavior of the lipid monolayer? is the rigidity onset point and the jammed state of the monolayer changed by addition of peptide? how is the final ‘collapse’ mode of relaxation modulated, does the monolayer fold, vesiculate, or crack? pressure/stress density/strain rigidity onset j a m i n g phase transitions relaxation via folding, lattice relaxation, lipid disks/stacks, etc. Our ultimate goal is to correlate some of the phenomena we see with the peptides to molecular models with the aid of high resolution NMR structures of the peptides.

Experimental Methods - FM and AFM Langmuir trough with fluorescence microscopy (FM) Lipid monolayer compressed at air-water interface -Use FM to image condensed domains which exclude fluorescent dye Atomic force microscopy (AFM) -Deposit lipid monolayer onto mica substrate for AFM analysis -AFM provides height information which is correlated to lipid phase

Effect on Monolayer Phase Behavior ∏=30mN/m DPPC:POPG 7:3 10% SPB 1-25 10% SPB 9-25 10% SPB 11-25 addition of peptide affects LC domain growth and increases the proportion of ‘bright’ phase on the surface entropy driven

de-jamming - peptide stress relaxation pathway A With no peptide the DPPC:POPG 7:3 system reaches a jammed state around 30-40mN/m; this is seen by the fact that the FM image derived structure factor S(q) does not change from low to high pressures. However addition of peptide (925, 1125, 125, 125Nflex) changes this behavior allowing for relaxation. S(q) for the systems with peptide develops nodes at 40-50mN that indicate a symmetry breaking transition, which can be seen on the FM imaging as LC domain banding. DPPC:POPG 7:3 SP-B 9-25 ∏= 30mN/m lattice packing rearrangement indicative of de-jamming and a novel mode of stress relaxation ∏= 50mN/m ∏= 70mN/m

Nanosilo formation - peptide stress relaxation pathway B Compression past the 45-55 mN/m plateau, FM imaging reveals speckles of increased intensity in the bright phase for SPB 1-25 and 9-25 due to an excess of material beneath the monolayer (lipid nanosilos JZ). Upon decompression, the fluorescent speckles gradually disappear hinting at a reversible respreading of the fluid lipid stored in the nanosilos, and allowing for complete recovery of monolayer post-collapse. 32.10mN/m 31.80mN/m 50s

Jamming and no nanosilos in pure DPPC:POPG 7:3 25mN/m 30mN/m -At higher pressures, there is a loss of disordered fluid phase (lower height) -FM bright phase associated with “fluid” region is as tightly packed as the condensed phase at higher pressure -At 70mN/m, monolayer is solid (‘jammed’) 60mN/m 70mN/m

Nanosilos and de-jamming of DPPC:POPG 7:3 w/ peptide SPB125 SPB925 AFM depositions done at 25C on a 3.18mPa·sec subphase at ∏=60mN/m Nanosilo size distributions: SPB125 SPB925 radius height 3, 9, 14nm 4nm 50-150nm 100nm even at high pressures (60mN/m) LC domains remain separated by disordered granular phase with all four peptides disordered granular phase allows for changes in S(q) and the new de-jamming stress relaxation pathway A LC domains: equal height (brighter, uniform) [dark phase on FM] disordered phase: granular with LC islands and lower (1nm) fluid phase coexisting with nanosilo stacks [bright phase on FM]

Nanosilos and de-jamming of DPPC:POPG 7:3 w/ peptide SPB125Nflex SPB1125 Nanosilo size distributions: 125Nflex SPB1125 radius height 2-3nm 3nm 35-40nm 50nm nanosilos also smeared on imaging indicating they were less stable tryptophan 9 plays a role in nanosilo stability the N-terminus clearly has some structural specificity due to the poly-P sequence that greatly enhances peptide mediated nanosilo formation nanosilo size: SPB125 >> SPB925, 125Nflex, 1125

So how does SP-B modulate the monolayer response FM/AFM conclusions So how does SP-B modulate the monolayer response to lateral stress and the mode of stress relaxation? phase behavior rigidity onset peptide un-jams the monolayer via  fluidity new-modes of relaxation opened collapse decreased phase separation two new modes of relaxation opened up by peptide addition A. nanosilos already seen by J Ding et al. are reconfirmed here for monomeric SP-B 125, we also show that the N-terminus plays a role in nanosilo formation. B. the symmetry-breaking transition allowed for by de-jamming seen in the monolayers containing peptide is a second form or releasing internal stresses not accessible to the pure DPPC:POPG 7:3 monolayer. relaxation pathways A and B likely allow the monolayer to release enough internal stresses such that the characteristic short  folding is not seen with the peptides

NMR Structural Studies We are hoping to understand on a molecular level the interactions of the peptide with the monolayer. A first step in this process is the solving of high-resolution solution structures of the peptides. facility: University of Chicago Nuclear Magnetic Resonance Facility NMR spectrometer: 600MHz Varian Inova with cold probe Experiments: All samples were prepared in CD3OH with 15mM DTT and 5mM peptide. Standard 2D homonuclear experiments were performed at 5oC including: TOCSY -> resonance assignments NOESY -> sequential assignments and interresidue restraints CTCOSY -> dihedral angle restraints Spectral analysis and sequential assignment of individual amino acid residues was performed manually with the aid of NMR Pipe and NMR View programs. Assignment of non-sequential NOESY crosspeaks was done automatically using ARIA. Structure calculation, refinement, and statistical analysis was performed with the ARIA platform using CNS as the molecular dynamics engine. Ramachandran and dihedral angle plots were generated using MolMol.

SBP 11-25 structure 15 backbone 2nd structure: 0.380858 ± 0.128222Å heavy atoms 2nd structure: 1.25356 ± 0.178359Å backbone all residues: 1.84112 ± 0.656818Å heavy atoms all residues: 2.32354± 0.508527Å mean RMSD 2 1 C R A L I K R I Q A M I P K G 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

SP-B 9-25 structure backbone 2nd structure: 1.64324 ± 0.468864Å 10 7 11 12 17 backbone 2nd structure: 1.64324 ± 0.468864Å heavy atoms 2nd structure: 2.73145 ± 0.452387Å backbone all residues: 2.0688 ± 0.441268Å heavy atoms all residues: 3.49047 ± 0.556568Å mean RMSD 3 2 1 W L C R A L I K R I Q A M I P K G 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Future Direction FM/AFM studies: NMR work: work at higher temperatures and different subphase viscosities and dielectric constants to attempt and reproduce physiological conditions potentially incorporate PA into the lipid components and repeat studies using advanced AFM techniques to obtain information about the elasticity and stiffness of different parts of monolayer and especially the nanosilos NMR work: continue work on refining the 9-25 structure, specifically trying to better define the residue 10 and 11 resonances. Also analyze and begin calculations on the methanol 1-25 structure. work on getting high resolution structures in dodecylphosphocholine micelle solutions that will provide a better lipid mimetic environment ultimately we hope to be able to obtain structures in lipid bicelle solutions in particular (DMPC:DHPC), this type of environment is most likely what the peptide is seeing when in the nanosilo stacks.

References and Funding Ding, J. et al. Nanostructure Changes in Lung Surfactant Monolayers Induced by Interactions between Palmitoyloleoylphosphatidylglycerol and Surfactant Protein B. Langmuir 19 (2003), 1539. Linge, JP et al. Assigning Ambiguous NOEs with ARIA. Methods in Enzymology 339 (2001), 71. Delaglio F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6 (1995), 277. Johnson, B. A., and Blevins, R.A. NMR View: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4 (1994), 603. Brunger AT et al. Crystallography and NMR System (CNS): A new software system for macromolecular structure determination. Acta Cryst. D 54 (1998), 905. Koradi, R. et al. MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graphics 14 (1996), 51. March of Dimes Birth Defects Foundation University of Chicago MRSEC

EXTRA FM/AFM conclusions phase behavior from the decreased LC area as well as the multi-height phases seen on AFM it is clear that the peptides increase monolayer fluidity in comparison to pure lipid and there does not seem to be a clear differentiation between the peptides on this basis leading us to believe the increase in fluidity is driven by entropy rigidity onset interestingly up to the first plateau seen with the peptides, the different monolayers have similar packing structures, however monolayers with the peptide undergo a symmetry breaking transition above the plateau indicating that the system is not jammed as in the pure lipid case collapse nanosilo formation can be seen as a form of collapse since clearly it is a path by which the monolayer is releasing internal stresses created by lateral compression So how does SP-B modulate the monolayer response to lateral stress and the mode of stress relaxation? The peptide opens up alternate routes of stress relaxation at lower lateral stress, which are not accessible to a pure lipid monolayer. We observe the formation of nanosilos, one mode of relaxation, and we also see the symmetry breaking packing transition that is a second form of stress release not seen in pure lipid monolayers. At this time it is difficult to say if there is a direct correlation between the two modes of relaxation. Symmetry breaking transitions have been observed in pure lipid systems (GM1:DPPC) hinting at the possibility that the two are not directly coupled. From the standpoint of the N-terminus, only the nanosilo formation seems to be affected by changing the peptide, again making it likely that the two new transitions are de-coupled.