Lipopolysaccharide (LPS), a component of Gram-negative bacterial outer membranes, comprises three regions: lipid A, core oligosaccharide, and O-antigen polysaccharide. Using the CHARMM36 lipid and carbohydrate force fields, we have constructed a model of an Escherichia coli R1 (core) O6 (antigen) LPS molecule. Several all-atom bilayers are built and simulated with lipid A only (LIPA) and varying lengths of 0 (LPS0), 5 (LPS5), and 10 (LPS10) O6 antigen repeating units; a single unit of O6 antigen contains five sugar residues. From (1)H,(1)H-NOESY experiments, cross-relaxation rates are obtained from an O-antigen polysaccharide sample. Although some experimental deviations are due to spin-diffusion, the remaining effective proton-proton distances show generally very good agreement between NMR experiments and molecular dynamics simulations. The simulation results show that increasing the LPS molecular length has an impact on LPS structure and dynamics and also on LPS bilayer properties. Terminal residues in a LPS bilayer are more flexible and extended along the membrane normal. As the core and O-antigen are added, per-lipid area increases and lipid bilayer order decreases. In addition, results from mixed LPS0/5 and LPS0/10 bilayer simulations show that the LPS O-antigen conformations at a higher concentration of LPS5 and LPS10 are more orthogonal to the membrane and less flexible. The O-antigen concentration of mixed LPS bilayers does not have a significant effect on per-lipid area and hydrophobic thickness. Analysis of ion and water penetration shows that water molecules can penetrate inside the inner core region, and hydration is critical to maintain the integrity of the bilayer structure.

Lipopolysaccharide, O-antigen, Escherichia coli, molecular dynamics, NMR spectroscopy, simulation, outer membrane

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,3,2	Ac	175.35	22.90
3,4,3	aDGalpN	99.80	50.76	68.02	78.22	73.27	61.85
3,4,2	bDGlcp	104.36	74.42	76.32	70.30	76.95	61.74
3,4	bDManp	101.38	78.57	77.14	67.57	77.47	61.10
3	bDManp	101.22	71.12	72.33	77.60	75.81	61.36
2	Ac	174.96	22.72
	aDGlcpN	99.08	53.69	81.40	68.98	72.56	60.95


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,3,2	Ac	-	2.05
3,4,3	aDGalpN	5.32	4.30	4.07	4.08	4.30	3.75-3.78
3,4,2	bDGlcp	4.68	3.39	3.52	3.43	3.43	3.73-3.86
3,4	bDManp	4.81	4.38	3.87	3.83	3.46	3.80-3.94
3	bDManp	4.79	3.98	3.83	3.83	3.56	3.74-3.89
2	Ac	-	2.11
	aDGlcpN	4.91	4.13	4.02	3.68	4.17	3.78-3.84


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,3,2	Ac		22.90/2.05
3,4,3	aDGalpN	99.80/5.32	50.76/4.30	68.02/4.07	78.22/4.08	73.27/4.30	61.85/3.75-3.78
3,4,2	bDGlcp	104.36/4.68	74.42/3.39	76.32/3.52	70.30/3.43	76.95/3.43	61.74/3.73-3.86
3,4	bDManp	101.38/4.81	78.57/4.38	77.14/3.87	67.57/3.83	77.47/3.46	61.10/3.80-3.94
3	bDManp	101.22/4.79	71.12/3.98	72.33/3.83	77.60/3.83	75.81/3.56	61.36/3.74-3.89
2	Ac		22.72/2.11
	aDGlcpN	99.08/4.91	53.69/4.13	81.40/4.02	68.98/3.68	72.56/4.17	60.95/3.78-3.84

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](O[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)[C@H](O[C@@H]3[C@@H](CO)O[C@@H](O[C@@H]4[C@@H](NC(C)=O)[C@@H](*)O[C@H](CO)[C@H]4O)[C@@H](O)[C@H]3O)O[C@H](CO)[C@H]2O)[C@H](NC(C)=O)[C@H]1O

892.811 g/mol (C₃₄H₅₆R₂N₂O₂₅, R = next and previous repeats, not counted in mol weight)