D e n i s e G r e a t h o u s e 1 , To d D . R o m o 2 , J o s h u a H o r n 2 , A l a n G r o s s f i e l d 2

1University of Arkansas, Fayetteville, AR, USA2University of Rochester Medical Center, Rochester, NY, USA

D e n i s e G r e a t h o u s e 1 , To d D . R o m o 2 , J o s h u a H o r n 2 , A l a n G r o s s f i e l d 2

1University of Arkansas, Fayetteville, AR, USA2University of Rochester Medical Center, Rochester, NY, USA

COMPARISON OF MEMBRANE INTERACTIONS OF ACYLATED AND NON-ACYLATEDLACTOFERRICINS BY SOLID-STATE NMR SPECTROSCOPY AND MOLECULAR DYNAMICS

COMPARISON OF MEMBRANE INTERACTIONS OF ACYLATED AND NON-ACYLATEDLACTOFERRICINS BY SOLID-STATE NMR SPECTROSCOPY AND MOLECULAR DYNAMICS

LfB6 (RRWQWR-NH2) is a tryptophan- and arginine-rich antimicrobial peptide with broad spectrum effectiveness derived from bovine lactoferrin. Membrane binding occurs via electrostatic interactions between arginines and negative charges on the bacterial cell membrane and intercalation of the tryptophans at the membrane inter-face. N-terminal acylation (CH3(CH2)4CO-RRWQWR-NH2; C6-LfB6) can enhance the antimicrobial activity (Greathouse et al. (2008) J. Pept. Sci 14:1103). Solid-state 2H and 31P NMR spectroscopy combined with all-atom and coarse-grained molecu-lar dynamics (CG-MD) simulations have confirmed subtle differences between 1:100 (peptide to lipid) LfB6 and C6-LfB6 in bilayers composed of 3:1 POPE:POPG (anionic, bacterial-like) and POPC (zwitterionic, mammalian-like). MD simulations reveal that the arginines of C6-LfB6 make first contact with POPE:POPG; whereas the C6 tails are first to contact POPC. LfB6 shows no sequence preference. Addi-tionally, C6-LfB6 inserts more deeply than LfB6 into both membranes. Tryptophan emission fluorescence spectra suggests the tryptophans in LfB6 and C6-LfB6 are more water exposed in neutral compared to anionic membranes, while CG-MD simulations reveal that LfB6 comes off the POPC membrane, exposing the trypto-phans to water. Acylation, therefore, increases the “stickiness” of the peptide for lipid bilayers. Although both peptides at 1:100 show significant membrane effects during short range simulations, C6-LfB6 has less influence on lipid order. We now compare experimental and molecular dynamics results for LfB6 and C6-LfB6 at 1:25 peptide to lipid. Solid-state 2H NMR spectra indicate that C6-LfB6 has a greater effect on the lipid acyl chain order at 1:25 compared to 1:100; whereas the effects of LfB6 are similar at both concentrations. Molecular dynamics simulations will be presented for comparison.

3:1 POPE:POPG• 8 peptides• 100 lipids per leaflet - POPE in green, POPG in blue• Solvated to 50% w/w (7,092 waters)• 50 mM salt (plus neutralizing)• ~48,000 atoms

POPC• 8 peptides• 90 lipids per leaflet - POPC in red• Solvated to 50% w/w (7,105 waters)• 50 mM salt (plus neutralizing)• ~47,000 atoms

• CHARMM 27 forcefield• Electrostatics using PME• 10 Å vdW cutoff• NPγT at 50ºC

• γ = 32.5 dyn/cm• 2 fs time step, RATTLE• NAMD on BlueGene• ~10.5 µs aggregate simulation time• All analyses performed using LOOS

Methods• Electron density along the membrane

normal• Membrane interface is defined as Z-axis

location of peak lipid head group density• Electron density for peptide backbone and

tryptophans plotted relative to membrane interface

• Density is normalized for visualization pur-poses

Discussion• Backbone is buried more deeply in POPC

than in POPE:POPG• Trp is more deeply buried in POPC• Trp density broadened for acylated peptide• Appears to contradict fluorescence data• Coarse-grained umbrella sampling indi-

cates peptide is less “sticky”

• LfB6 and C6-LfB6 peptides have minimal effects on the lipid head groups of neutral and anionic lipids by 31P NMR

• 2H NMR Spectra reveal that: - Both peptides have little effect on the

order of the terminal methyl groups of POPC, POPE, or POPG at 1:100 or 1:25

- Both peptides decrease the order of all lipids in vicinity of head groups, except for LfB6 in POPC which increases the order

- The effects on lipid order for C6-LfB6 at 1:100 and 1:25 are similar

- C6-LfB6 results in larger decrease in order at 1:25 compared to 1:100

- Broadening of POPE-d31 2H-line widths is observed in the presence of LfB6, but not C6-LfB6

• Simulations show same trend in membrane order as experiment for 1:25

- Reduced order is a short-range effect• Trp residues in both peptides are more

water-exposed in neutral POPC membranes than in anionic mixture POPE:POPG

• Coarse-grained umbrella sampling indicates peptides associate less tightly with POPC membranes than POPE:POPG despite going deeper when bound

• Umbrella sampling with coarse-grained MD gives ∆∆G estimates for POPE:POPG vs POPC:

-2.9 kcal/mol (C6-LfB6) -2.8 kcal/mol (LfB6)• Estimated ∆∆G from acylation: -1.5 kcal/mol (POPC) -1.7 kcal/mol (POPE:POPG)

Acylated Lactoferricin Peptide

CholesterolZwitterionicPhospholipid

AnionicPhospholipid

Hydrophobic InteractionsElectrostatic and

Hydrophobic InteractionsW

eakStrong

POPC-d31 (neutral)

HDDD

D D

D D

DD

DD DD

D D

D D

D D

D D

D D

D D

D DO

DD

D D

O

O

O

PO-

O

O

NH3+O

POPE-d31 (neutral)

HDDD

D D

D D

DD

DD DD

D D

D D

D D

D D

D D

D D

D DO

DD

D D

O

O

O

PO-

O

O

O OHOH

Na+

POPG-d31 (anionic)LfB Peptides

1 kcal/mol

5 80604020Temperature, ºC

POPE:POPG (3:1)

Distance from Membrane Surface (Å)

No

rmal

ize

d E

lect

ron

De

nsi

ty

Backbone

Trp

Backbone

Trp

PE:PG (C6LfB) PE:PG (LfB) PC (C6LfB) PC (LfB)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

−15 −10 −5 0 5 10 15 −15 −10 −5 0 5 10 15

POPC POPE:POPG (3:1)

C6-LfB

LfB

Lipid Alone

Multi-Lamellar Vesicles1:50 Peptide:Lipid

+H3N-Arg-Arg-Trp-Gln-Trp-Arg-Amide

CH3(CH2)4CO-Arg-Arg-Trp-Gln-Trp-Arg-AmideLfB Peptides

POPE POPGC6LfB

C6

ArgTrpGln

C6LfB

C6

ArgTrpGln

POPC

Discussion• Minimal binding for LfB and C6-

LfB6 in neutral lipid (POPC)• Acylation enhances binding• Even 10% DOPG (anionic) in-

creases binding• DOPE:DOPG (3:1) 80-100%

bound

MLVs, 1:50,10 mM NaCl10 mM Tris, pH 7.5

0

40

80

120

160

LfB6 C6-LfB6

E. coliS. aureus

MIC

(mg/

ml)

Peptide

0

25

50

75

100

0.1% Triton LfB6 C6-LfB6

% H

emol

ysis

100 g/mlControl

0

25.0

50.0

75.0

100.0

LfB Native C6 LfB native

DOPC DOPC:DOPG (90:10) DOPE:DOPG (75:25)

Axi

s

Axis

= 0 = 90

1. Apply peptide/lipid mixture

2. Hydrate (50%) and stack

3. Place in cuvette, cap, and seal

4. Place oriented sample in NMR probe at = 0º or 90º

40 0 -40kHz

POPCPOPC-d31

POPE-POPG (3:1)POPE-d31POPG-d31

40 0 -40

kHz

40 0 -40kHz

1:25 1:25

1:100 1:100

40 0 -40

kHz

Lipid Only

+ LfB6

+ C6-LfB6

Lipid Only

+ LfB6

+ C6-LfB6

40 0 -40kHz

40 0 -40kHz

1:1001:100 1:251:25Minimum (kHz) Maximum (kHz) Minimum (kHz) Maximum (kHz)

POPC-d31 5.6 55.8 4.4 47.2 + LfB Native 5.2 (-0.4) 52.8 (-3.0) 4.0 (-0.4) 44.2 (-3.0)

+ C6-LfB Native 5.5 (-0.1) 54.8 (-1.0) 4.0 (-0.4) 45.0 (-2.0)

POPE:POPG-d31 5.4 54.2 5.7 56 + LfB Native 5.1 (-0.3) 52.1 (-2.1) 5.3 (-0.4) 53 (-3.0)

+ C6-LfB Native 5.3 (-0.1) 53.9 (-0.3) 5.3 (-0.4) 54.5 (-1.5)

POPE-d31:POPG 5.2 54.0 5.7 56.4 + LfB Native 5.4 (+0.2) 55.1 (+1.1) 5.7 (0) 56.9 (+0.5)

+ C6-LfB Native 5.3 (+0.1) 53.8 (-0.2) 5.3 (-0.3) 53.9 (-2.5)

Changes in Minimum and MaximumQuadrupolar Splittings ( q) for LfB Peptides

-4

-2.9

-1.8

-0.7

0.4

1.5

POPC-d31POPG-d31POPE-d31

LfB6 C6-LfB6

Maximum q (kHz)

-4

-2.9

-1.8

-0.7

0.4

1.5

POPC-d31POPG-d31POPE-d31

LfB6 C6-LfB6

Minimum q (kHz)

1:100 1:25 1:100 1:25 1:100 1:25 1:100 1:25

Discussion• LfB6: Slight increase in Tm

• C6-LfB6: No change in Tm

Discussion• Minimal changes in 31P CSA for POPC or POPE:POPG in presence of LfB6 or C6LfB6 (1:50)

Discussion• Little change in minimum quadrupolar splittings for either peptide at 1:25 or 1:100• Larger changes in maximum splittings compared to minima for both peptides • Changes greater at 1:25 compared to 1:100 for C6-LfB6

Discussion• Little hemolytic activity• C6-LfB6 slightly more hemolytic than LfB6

Discussion• Spectral shift indicates

water exposure of Trp

• POPC: - LfB6 > C6-LfB6

• POPE:POPG - Less than POPC - LfB6 > C6-LfB6

300 320 340 360 380 400 420 4400.0

0.2

0.4

0.6

0.8

1.0

LfB6 vs C6-LfB6,POPC and POPE:POPG, 1:50

Nor

mal

ized

Flu

ores

cenc

e In

tens

ity, a

.u.

Emission, nm

LfB6 POPC (352 nm)LfB POPE:POPG (339/342 nm)C6-LfB6 POPC (345 nm)C6-LfB6 POPE:POPG (339 nm)

POPE Tm 25º CTh 71º C

POPE:POPG, 20.5º C

LfB6 + POPE:POPG, 21.4º C

C6-LfB6 + POPE:POPG, 20.4º C

hRBCs

% P

eptid

e Bo

und

40 0 -40 ppm 40 0 -40 ppm

(∆∆ν

q) M

ax (k

Hz)

(∆∆ν

q) M

in (k

Hz)

Methods• Acyl C-H bond orientation relative

to membrane normal• Experimentally measured by deute-

rium quadrupolar splitting in solid state NMR

• δSCD is the difference in order pa-rameter between the membrane with peptide and the neat mem-brane

Discussion• Order decreased for POPC by both

C6-LfB6 and LfB6• Order decreased for POPE and

POPG by C6-LfB6• Order increased for POPE by C6-

LfB6, but POPG is decreased

C6-

LfB

6 (1

:25)

C6-

LfB

6 (1

:25)

LfB

6 (1

:25)

LfB

6 (1

:25)

HP

O-O

O

N+ODD

DD D

D D

DD

DD DD

D D

D D

D D

D D

D D

D D

D DO

DD

D D

O

O

O

AbstractAbstract

ConclusionsConclusions

Experimental MethodsExperimental Methods

Experimental ResultsExperimental Results

Molecular DynamicsMolecular Dynamics

Molecular DynamicsMolecular Dynamics

Membrane Order ParametersMembrane Order Parameters

Lightweight Object Oriented Structure Analysis

G ro s s f ie l d L a b

LOOS (Lightweight Object Oriented Structure analysis library) is an open-source library for analyzing molecular simulations from the Grossfield Lab.

Scan the QR code for a PDF copy of this poster

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informationabout LOOS

The NMR facilities are supported by NIH GM1034501 and GM103429. Funding was also provided by NSF MCB 0841227 and the Arkansas Biosciences Institute. AG funding provided by NIH GM095496. We thank the Center for Integrated Research Computing at the University of Rochester for providing the BlueGene.

Simulation DetailsSimulation Details

System ConstructionSystem Construction

Water Exposure (Electron Density)Water Exposure (Electron Density)1:25 1:1001:25 1:100

31P NMR31P NMR

Minimum Inhibitory ConcentrationMinimum Inhibitory Concentration Hemolytic AssayHemolytic Assay

Differential Scanning CalorimetryDifferential Scanning Calorimetry

Trp Fluorescence SpectroscopyTrp Fluorescence Spectroscopy

Partitioning AssaysPartitioning Assays

2H NMR2H NMR

loos.sourceforge.nett

inyurl.com/lfb2013

δSCD

δSCD

Acyl Carbon Acyl Carbon

-0.015

-0.01

-0.005

0

0.005

0.01

2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16

-0.015

-0.01

-0.005

0

0.005

0.01

2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16

POPE (Sim)POPE (Exp)

POPG (Sim)POPG (Exp) POPC (Sim) POPC (Exp)