Water Interactions with Membrane Proteins & Other ... Lect… · Water is Important for Most...
Transcript of 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
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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
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
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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
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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
• 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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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
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
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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.
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
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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
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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.
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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