water interactions with membrane proteins & other ... lect… · water is important for most...

Water Interactions with Membrane Proteins & Other Biomolecules from

1H-X Heteronuclear Correlation NMR

4th Winter School on Biomolecular Solid-State NMR, Stowe, VT, Jan. 10-15, 2016

Mei Hong Department of Chemistry, MIT

•  Diversity of water interactions with biomolecules

•  SSNMR techniques: spin diffusion, HETCOR, & dipolar-dephased HETCOR

•  Mechanism of water 1H transfer: chemical exchange & spin diffusion

•  Water for studying ion channels •  Open & closed states •  TM helix structure •  Site-specific hydrogen bonding

•  Hydration and H-bonding of Arg residues in antimicrobial peptides •  Dehydration & curvature induction of membranes by viral fusion proteins

•  Water interactions with plant cell wall polysaccharides

•  Water dynamics at low T: effects of cryoprotectants on membrane structure

Outline

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Water is Important for Most Biological Systems

•  Ion conduction & transport in ion channels •  Hydrogen bonding and charge distribution •  Hydration of polar & charged residues in proteins & carbohydrates •  Hydration & dehydration of membrane surfaces for function •  Low-temperature behavior of water: ice formation & glass formation 3

Water - Protein Heteronuclear Correlation NMR

Williams & Hong, JMR, 2014. 4

Heterogeneous Water Dynamics of Hydrated Lipid Membranes

•  Water dynamics of various lipid membranes: POPC/cholesterol ≤ POPE < POPG

•  "Water" 1H T2 is the average T2 of water & labile protons.

•  Bulk water •  Inter-lamellar water on the membrane surface •  Water in transmembrane channels

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Mechanism of Water → Protein 1H Polarization Transfer: Chemical Exchange & Spin Diffusion

Doherty & Hong, JMR, 2008. 6

•  Diversity of water interactions with biomolecules

•  SSNMR techniques: spin diffusion, HETCOR, & dipolar-dephased HETCOR

•  Mechanism of water 1H transfer: chemical exchange & spin diffusion

•  Water for studying ion channels •  Open & closed states •  TM helix structure •  Site-specific hydrogen bonding

•  Hydration and H-bonding of Arg residues in antimicrobial peptides •  Dehydration & curvature induction of membranes by viral fusion proteins

•  Water interactions with plant cell wall polysaccharides

•  Water dynamics at low T: effects of cryoprotectants on membrane structure

Outline

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Proton Conduction by the Influenza M2 Channel

?

Water protons M2 protons

Exchange & Spin diffusion

M2 13C spins

Low pH High pH

Khurana et al, PNAS, 106, 1069 (2009).

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Water-M2 Interaction in Lipid Membranes

Luo & Hong, JACS, 2010. 9

1H Polarization Transfer: Water-Protein Surface Area DQ detection to suppress lipid background 13C signals

IP (tm )IP (∞)

≈Deff

πSWPVP

tmLuo & Hong, JACS, 2010.

10

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M x,y ,z(tm + Δtm ) = M x,y ,z(tm ) +DijΔtm

a2i∑ Mi (tm ) −M x,y ,z(tm )[ ]

3D Lattice Simulations of Spin Diffusion

•  Vprot = 12.7 nm3 (ρ=1.43 g/cm3). •  VAmt = 0.2 nm3.

•  Helix tilt: 20˚-30˚. •  DWP = 0.008 nm3/ms. •  Indirect W–>L–>P pathway ignored.

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Model of the Water-Filled Pore: Drug Dehydrates the Channel

pH 7.5 + Amt

Luo & Hong, JACS, 2010. 12

Water-Exposed Surface Area of M2 Changes with Channel Opening and Drug Binding

Luo & Hong, JACS, 2010. 13

Periodicity in TM Helix Bundle Structure from Water 1H Polarization Transfer

14

Lipid- vs Pore-Facing Residues & pH-Dependent Channel Diameters from Water Transfer Profiles

Williams & Hong, JMR, 2014. 15

Helical Periodicity in Water → Protein 1H Polarization Transfer Profile

Williams & Hong, JMR, 2014. 16

Proton Conduction Mechanism in M2

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1H Transfer Between Water & His37 From HETCOR

RN ...O = 2.63 Å

Hong et al, JACS, 2012. 18

•  Diversity of water interactions with biomolecules

•  SSNMR techniques: spin diffusion, HETCOR, & dipolar-dephased HETCOR

•  Mechanism of water 1H transfer: chemical exchange & spin diffusion

•  Water for studying ion channels •  Open & closed states •  TM helix structure •  Site-specific hydrogen bonding

•  Hydration and H-bonding of Arg residues in antimicrobial peptides •  Dehydration & curvature induction of membranes by viral fusion proteins

•  Water interactions with plant cell wall polysaccharides

•  Water dynamics at low T: effects of cryoprotectants on membrane structure

Outline

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Extensive 13C-31P REDOR distance data revealed the existence of Arg-phosphate salt bridges in PG-1 and other cationic AMPs:

Stabilized by: •  electrostatic attraction •  hydrogen bonding

Cationic Antimicrobial Peptides: Arginine-Phosphate Salt Bridges

Protegrin-1

Qu et al, Infect. Immun. 64, 1240 (1996).

Do water molecules play a role in stabilizing Arg-phosphate salt bridges?

oligomeric structure: β-barrel in bacterial membranes

20

HETCOR Spectra of an Antimicrobial Peptide

Regular 1H-13C & 1H-15N HETCOR spectra do not allow unambiguous distinction of Hα and water protons (4.5-5 ppm).

PG-1 in POPE/POPG membrane, 283 K

Li et al, J. Phys. Chem, 2010. 21

13C- and 15N Dipolar Dephasing in HETCOR: Distinguish Organic Protons from Water Protons

Yao et al, JMR, 2001. 22

13C, 15N MELODI-HETCOR Allows

Assignment of Water •  HN and guanidinium 1H’s assigned

by 15N MELODI. •  Hα (~4.8 ppm) assigned by 13C

MELODI. •  2 ms HH-CP sufficient to detect

water cross peak to guanidinium.

Membrane-bound Arg is solvated by water.

Li et al, J. Phys. Chem, 2010. 23

MD Simulations of Arg-Water Interactions in HIV Tat Tat: GRKKR RQRRR PPQ. A cationic cell-penetrating peptide.

Herce & Garcia, PNAS, 2007. 24

•  Diversity of water interactions with biomolecules

•  SSNMR techniques: spin diffusion, HETCOR, & dipolar-dephased HETCOR

•  Mechanism of water 1H transfer: chemical exchange & spin diffusion

•  Water for studying ion channels •  Open & closed states •  TM helix structure •  Site-specific hydrogen bonding

•  Hydration and H-bonding of Arg residues in antimicrobial peptides •  Dehydration & curvature induction of membranes by viral fusion proteins

•  Water interactions with plant cell wall polysaccharides

•  Water dynamics at low T: effects of cryoprotectants on membrane structure

Outline

25

Virus-Cell Membrane Fusion

•  High membrane curvature. •  Partial dehydration of the membrane surface.

Viral fusion requires

26

What is the Conformation of the Transmembrane Domain of the

Parainfluenza Virus?

27

TMD Conformation Also Depends on the Lipids

Yao et al. PNAS, 2015. 28

Strand-Helix-Strand in PE Membranes

Helicity = ICαβ/CαC’, helix/(ICαβ/CαC’,helix + ICαβ/CαC’,strand)

Spo

ntan

eous

cur

vatu

re

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The Viral TMD Dehydrates PE Membranes

Yao et al. PNAS, 2015.

The β-strand TMD reduces the % of membrane-bound water and increases bulk water %: the peptide dehydrates PE-rich membranes.

1H-31P HETCOR

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TMD Induces Strong Curvature to PE Membranes

TMD quantitatively converts the DOPE spectrum to an isotropic peak.

+ TMD DOPE

31P chemical shift (ppm) 31P chemical shift (ppm)

SAXS: TMD Induces an Ia3d Phase to DOPE

•  An Ia3d cubic phase coexists with an HII phase.

•  Q-ratios for the Ia3d phase gives the lattice parameter, which suggests a hemifusion stalk with a “waist” of 10 nm, similar to the pure-DOPE stalk waist of ~ 9 nm. (Siegel, 1999).

Gerard Wong, UCLA

Yao et al. PNAS, 2015.

Ia3d (gyroid)

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TMD Uses the β-Sheet Conformation to Induce NGC & Stabilize Hemifusion Intermediates

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•  β-strands are anisotropic and can have different surface orientations, which can cause different curvatures to the two membrane leaflets.

•  The NGC is characteristic of hemifusion intermediates.

•  The β-strand is likely oligomerized into β-sheets.

Yao et al. PNAS, 2015.

•  Diversity of water interactions with biomolecules

•  SSNMR techniques: spin diffusion, HETCOR, & dipolar-dephased HETCOR

•  Mechanism of water 1H transfer: chemical exchange & spin diffusion

•  Water for studying ion channels •  Open & closed states •  TM helix structure •  Site-specific hydrogen bonding

•  Hydration and H-bonding of Arg residues in antimicrobial peptides •  Dehydration & curvature induction of membranes by viral fusion proteins

•  Water interactions with plant cell wall polysaccharides

•  Water dynamics at low T: effects of cryoprotectants on membrane structure

Outline

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Plant Cell Walls: A Carbohydrate-Protein Complex

•  Provide rigidity to plant cells. •  Regulate plant growth. •  Store energy as carbohydrates.

Cellulose microfibril

Plant cell walls

What is the 3D structural arrangement of polysaccharides in the cell wall?

Interior crystalline cellulose

Surface cellulose

35

Polygalacturonic acid (-)

Carbohydrate Structure in Plant Cell Walls

36

Results from 2D & 3D MAS Spectra: Single-Network Model of the Plant Cell Wall

• Cellulose, hemicellulose, and pectins coexist in a single 3D network instead of two separate networks (13C cross peak data)

• Hemicellulose does not coat the cellulose microfibril surface, but is embedded into the microfibril at limited spots.

•  Pectins: one fraction binds cellulose and is immobilized, while another fraction is interstitial and highly dynamic.

37 Dick-Perez et al, Biochemistry, 2011.

Water Dynamics is Sensitive to the Polysaccharide Content of the Cell Wall

White…Cosgrove & Hong, JACS, 2014. 38

Water-Polysaccharide 1H Transfer: Buildup Curves

Water-cellulose transfer lags behind water-matrix transfer.

White…Cosgrove & Hong, JACS, 2014. 39

Ca2+ crosslinked wall, more bound water, fast 1H SD

Ca2+-depleted CW, dynamic water, slow 1H SD

Sparse cell wall after extraction, fast 1H SD rate.

Charge & Pore Size Affect Water Dynamics

40

2D HETCOR Reveals Dynamic Differences Between Bound & Bulk Water in Cell Walls

White…Cosgrove & Hong, JACS, 2014. 41

•  Diversity of water interactions with biomolecules

•  SSNMR techniques: spin diffusion, HETCOR, & dipolar-dephased HETCOR

•  Mechanism of water 1H transfer: chemical exchange & spin diffusion

•  Water for studying ion channels •  Open & closed states •  TM helix structure •  Site-specific hydrogen bonding

•  Hydration and H-bonding of Arg residues in antimicrobial peptides •  Dehydration & curvature induction of membranes by viral fusion proteins

•  Water interactions with plant cell wall polysaccharides

•  Water dynamics at low T: effects of cryoprotectants on membrane structure

Outline

42

Cryoprotection of Lipid Membranes for Low-T NMR

Phase diagrams

glycerol DMF

Murata and Tanaka, Nat. Materials, 2012 Baudot & Boutron, Cryobiology, 1998. 43

Glycerol: Limited Cryoprotective Ability

44 Lee & Hong, J. Biomol. NMR, 2014.

DMSO: Excellent Cryoprotection Down to 200 K

45 Lee & Hong, J. Biomol. NMR, 2014.

46

Trehalose < Glycerol << PEG < DMF < DMSO

Lee & Hong, J. Biomol. NMR, 2014.

47

T-Dependent 13C Linewidths: DMSO Orders the Glycerol Backbone & Chain Termini

Lee & Hong, J. Biomol. NMR, 2014.

DMSO Depth & Immobilization of Lipids

The less mobile the lipid is at high T (e.g. by DMSO binding), the more ordered it is at low T.

β

α

48

Summary

•  Ion channel structure and dynamics; •  TM helix protein topology; •  H-bonding to Arg to lower the ΔG of insertion of cationic membrane peptides;

•  Membrane dehydration by viral fusion proteins; •  Water dynamics in complex biomaterials such as plant cell walls.

1H-13C, 1H-15N, and 1H-31P HETCOR with optional dipolar dephasing is a versatile approach for studying water interactions in many biomolecules:

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MIT & ISU Lipid hydration: Tim Doherty

M2: Wenbin Luo, Jonathan Williams, Keith Fritzsching

AMP: Shenhui Li, Ming Tang

Viral fusion: Hongwei Yao, Yu Yang, Michelle Lee

Plant cell walls: Tuo Wang, Paul White

Low-T NMR: Myungwoon Lee

Collaborators Prof. Bill DeGrado, Jun Wang & Yibing Wu (UCSF) Prof. Alan Waring (UCLA) Prof. Gerard Wong (UCLA) Prof. Daniel Cosgrove (Penn State) Prof. Olga Zabotina (ISU)

Acknowledgement

Funding

2013

2015