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








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.

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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


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


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

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


Helical Periodicity in Water → Protein 1H Polarization Transfer Profile


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








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

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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








Outline

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Virus-Cell Membrane Fusion

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

Viral fusion requires

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What is the Conformation of the Transmembrane Domain of the

Parainfluenza Virus?

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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


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.









Outline

34

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

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Polygalacturonic acid (-)

Carbohydrate Structure in Plant Cell Walls

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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.


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

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









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.

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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

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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

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

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