Correlating Gramicidin IonChannel Formation to Artificial 

Membrane Dynamics

Temiloluwa Okusolubo

University of Maryland, Baltimore County, Department of Biological Sciences

Mentors: Dr. Michihiro Nagao and Dr. Elizabeth Kelley
Lipid Membranes

Cell membranes contain an equal 

ratio of proteins to lipids. Lipid-lipid 

ratios are rigidly maintained. 

Lipid and protein composition 

determines membrane structure and 

dynamics.

Cell function and disease have a direct 

link to nanoscale membrane dynamics 

and macroscopic structure

http://lipidbuilder.epfl.ch/img/membrane.png
Gramicidin

Gramicidin channels provide a unique 

combination of advantages that sets 

them apart from other channels.

- Structure of the bilayer-spanning 

channel is known 

- It’s ion permeability is well known and 

can be modified

- Lipid-protein interaction is universal in 

nature. 

Beaven et al. Gramicidin A Channel Formation Induces Local Lipid Redistribution I: 

Experiment and Simulation
Lipid-Protein Vesicles Were Made via Extrusion 

https://avantilipids.com/divisions/equipment-products

100 nm

Hydrophilic heads 

Hydrophobic tails 

100 nm

https://www.wur.nl/en/show/MSc-Cell-membranes-breaking-the-barrier-using-nanoparticles-1.htm

https://en.wikipedia.org/wiki/Lipid_bilayer#/media/File:Phospholipids_aqueous_solution_structures.svg
Density

Partial specific volume (vs) was determined from measurements taken of the 

lipid solution density by the following equation:

vs = 

1

ρρ0

(1 -

ρρss −ρρ0

cc ) 

Volume per lipid molecule (VL) was determined from measurements taken of 

the lipid solution density by the following equation:

VL = 

vs

NNAA

∑ xxii MMii

DLPC

A 12-C Lipid

DMPC

A 14-C Lipid

1,2-dilauroyl-sn-glycero-3-phosphocholine 1,2-dimyristoyl-sn-glycero-3-phosphocholine

HYDROPHOBIC 

MISMATCH

https://ac.els-cdn.com/S0005273607001848/1-s2.0-S0005273607001848-main.pdf?_tid=b0f57ec4-9014-4a1f-9c21-

dbea993914cf&acdnat=1533184309_7906058a99389690d5d5c648b174e460
DLPC

9.8E-22

9.9E-22

1E-21

1.01E-21

1.02E-21

1.03E-21

1.04E-21

0 10 20 30 40 50 60

Volume per Lipid Molecule (mL)

Temperature (°C)

Volume per Lipid Molecule of DLPC

pure DLPC

0.25% G + DLPC

0.76% G + DLPC

1.25% G + DLPC
DMPC

9.6E-22

9.8E-22

1E-21

1.02E-21

1.04E-21

1.06E-21

1.08E-21

1.1E-21

1.12E-21

0 10 20 30 40 50

Volume per Lipid Molecule (mL)

Temperature (°C)

Volume per Lipid Molecule of DMPC

pure DMPC

0.25% G + DMPC

0.76% G + DMPC

1.25% G + DMPC

Tm = 24 °C

https://phys.libretexts.org/LibreTexts/University_of_California_Davis/UCD%3A_Biophysics_241_-

_Membrane_Biology/Membrane_Phases/The_Gel_Phase
Gramicidin Conformation in 1,2-dilauroyl-snglycero-3-phosphocholine (DLPC)

-50

-40

-30

-20

-10

0

10

20

30

40

50

200 220 240 260 280

Difference in Absorbance of Polarized Light 

(millidegrees)

Wavelength (nm)

Circular Dichroism: Gramicidin β-helical Structure

0.25% G

0.76% G

1.25% G

Koeppe and Andersen, Annu. Rev. Biophys. Biomol. Struct. 1996, 25: 231-258

channel forming 

non channel forming
Dynamic Light Scattering (DLS)

A technique used to measure the 

hydrodynamic radius of nanoparticles 

suspended in solution. 

Particle size can be determined by 

measuring the random changes in the 

intensity of light scattered from a 

suspension or solution.

Used to determine the size of our 

artificial membranes
0

10

20

30

40

50

60

70

80

05 10 15 20 25 30

Average Radius for 50X Dilution (nm)

Days After Extrusion

Change in DLPC Sample Radii Over Time

pure DLPC

0.25% DLPC

0.76% DLPC

1.25% DLPC
Small Angle Neutron Scattering (SANS)

Information about membrane 

structure is gleaned from 

contrast between deuterated 

lipid tails and solvent compared 

to head groups.

Y.A. Hassan, E.E. Dominguez-Ontiveros / Nuclear Engineering and 

Design 238 (2008) 3080–3085

h-lipid

d-lipid
0.01

0.1

1

10

100

0.005 0.05 0.5

Intensity (cm-1)

Q (A-1)

Raw SANS Data for Deuterated DLPC 

Samples (20°C) 

0.06

0.6

0.05 0.5

Intensity (cm-1)

Q (A-1)

pure DLPC (20C)

0.25 G + DLPC

0.76% G + DLPC

1.25% G + DLPC

shell

shell

core
https://ac.els-cdn.com/S0005273607001848/1-s2.0-S0005273607001848-main.pdf?_tid=b0f57ec4-9014-4a1f-9c21-

dbea993914cf&acdnat=1533184309_7906058a99389690d5d5c648b174e460

38

39

40

41

42

43

44

45

46

15 25 35 45 55

Membrane Thickness (Å)

Temperature (°C)

Membrane Thickness Decreases with Increasing 

Temperature

pure DLPC

0.25% G + DLPC

0.76% G+ DLPC

1.25% G + DLPC
height fluctuations 

in the membrane

https://upload.wikimedia.org/wikipedia/commons/c/c6/Phospholipids_aqueous_solution_structures.svg
Neutron Spin Echo (NSE)

- Takes advantage of a neutrons Larmor Precession

- Differences in precession are analyzed 

- Basic information about the structure and dynamics of 

the matter 

https://neutrons.ornl.gov/sites/default/files/Zolnierczuk_Introduction_to_NSE2.pdf
II(qq,tt)

II(qq, 0)

= exp[ − (ΓZZZZtt)

2

3]

Where:

ΓZZZZ= relaxation rate

t = Fourier time

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

0 20 40 60 80 100 120

I(q,t)/I(q,0)

Fourier Time (ns)

q-t correlation for pure DLPC

q=0.039 1/A

q=0.049 1/A

q=0.053 1/A

q=0.063 1/A

q=0.073 1/A

q=0.08 1/A

q=0.094 1/A

q=0.11 1/A
RELAXATION RATE

Collective height fluctuations can be used to quantify membrane 

elastic bending modulus from NSE experiments with the following 

equation:

ΓΓZG = 0.0069

kkBTT

kk

kkBBTT

ηη

QQ3

Where:

ΓΓZG = relaxation rate

kkB = Boltzmann constant

kk= Bending modulus

ηη= solvent viscosity

height fluctuations 

in the membrane
Membrane Elasticity Increases 

Lee et. al. Phys. Rev. Letter, 2010, 038101

NSE Results are Similar to Previously Conducted Experiments
Contrasting Trends

VS.

43

43.5

44

44.5

45

45.5

Membrane Thickness (Å)

0 0.5 1 1.5

mol% Gramicidin

Membrane Thickness Decreases with 

Increasing mol% Gramicidin

Membrane Elasticity Increases 

with Increasing mol% Gramicidin 
FUTURE WORK

We can solve for the Area Compressibility Modulus (KA) with the Thin Sheet Theory:

KA =

βKK

ddtt

2

Where:

KKAA= area compressibility modulus

β = coupling constant between membrane 

leaflets

KK = bbbbbb bbbb mmmm mmmmmmmm

ddtt = mm mm mmmm ttt tt
ACKNOWLEDGEMENTS

This investigation was sponsored by NIH/NIGMS MARC U*STAR T34 HHS 00001 National Research Service Award 

to UMBC 

- Dr. Michihiro Nagao and Dr. Elizabeth Kelley

- Dr. Michael Zhang

- Dr. Joseph Dura and Dr. Julie Borchers

- NIST SURF director Dr. Brandi Toliver

- NCNR staff

- My fellow SURFers

- Center for High Resolution Neutron Scattering
FUTURE WORK

We can solve for the Area Compressibility Modulus (KA) in two 

ways:

Thin Sheet Theory

KA =

βKK

ddtt

2

Statistical Mechanics

KA =

kkBBTT

σσAA

2AA0

Which can be combined as :

K =

kkBBTT

βσσAA

2

ddtt

2

AA0

Where:

ddtt = mm mm mmmm ttt tt

AA00 = ll h

= 

VVLL

ddtt

β = coupling constant 

between membrane leaflets

kkBB = BB BB CC

σσAA = fractional area change

KK = bbbbbb bbbb mmmm mmmmmmmm