1. 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

2. 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

3. 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.

4. 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.

5. 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

6. 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

7. 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

8. Nanosilo formation - peptide stress relaxation pathway B
Compression past the 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

9. 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

10. 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]

11. 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

12. 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

13. 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.

14. SBP 11-25 structure 15 backbone 2nd structure: 0.380858 ± 0.128222Å
heavy atoms 2nd structure: ± Å
backbone all residues: ± Å
heavy atoms all residues: ± Å
mean
RMSD 2 1
C R A L I K R I Q A M I P K G

15. SP-B 9-25 structure backbone 2nd structure: 1.64324 ± 0.468864Å10 7 11 12 17
backbone 2nd structure: ± Å
heavy atoms 2nd structure: ± Å
backbone all residues: ± Å
heavy atoms all residues: ± Å
mean
RMSD 3 2 1
W L C R A L I K R I Q A M I P K G

16. 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.

17. 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

18. 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.