Mul$dimensional  correla$on  techniques  for  (membrane)  protein  

resonance  assignments  and  structure  determina$on  

 Vlad  Ladizhansky    

University  of  Guelph,    Ontario,  Canada  

•  Microcrystalline proteins/

• protein complexes

•  Amyloids

•  Membrane proteins

•  Cell walls, biomaterials

•  Molecular systems in situ

Spectroscopic MethodsDOI: 10.1002/anie.201002823

Solid-State NMR Spectroscopy on ComplexBiomoleculesMarie Renault, Abhishek Cukkemane, and Marc Baldus*

AngewandteChemie

Keywords:amyloid · biomolecules ·magic-angle spinning ·membrane proteins ·NMR spectroscopy

M. Baldus et al.Reviews

8346 www.angewandte.org ! 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2010, 49, 8346 – 8357

S.  Wang  &  V.  Ladizhansky  (2014)  Prog.  Nucl.  Magn.  Res.  Spec.  

OUTLINE 1.  Brief  comments  on  sample  preparaMon  of  membrane  proteins                  2.  Two-­‐dimensional  experiment:  an  example  of  a  2D  13C-­‐13C  correlaMon    

 spectrum    3.  13C-­‐detected  NMR:    methods  for  spectroscopic  assignments    4.  Structure  determinaMon  

Proteins used as examples in this talk

Anabaena  Sensory  Rhodopsin  (ASR)  Light  sensor,  229  aa  

229  aa  

C1284H1916N316O318S11

Proteorhodopsin  (PR)    Proton  pump,  251  aa  

C1334H1986N312O347S13

Human  Aquaporin  1  Water  channel,    282aa    

C1406H2228N382O407S7

Sample preparation of membrane proteins

Samples: E. coli-expressed, His-tag purified, reconstituted in DMPC:DMPA (9:1) at a protein/lipid ratio 2:1 (w/w). (protocol and pictures by I. Kawamura)

Overexpressed   Solubilized   Mixed  with  Lipids  and  Bio-­‐beads  

Recons$tuted   Packed  

The Utility of FTIR Spectroscopy in NMR Sample Preparation

•  Absolute Spectra (A): 1.  Quantity of protein 2.  Exact lipid/protein ratio 3.  Nativity of the secondary

structure 4.  Extent of the isotope

labeling

•  Light-Induced Difference Spectra (B):

1.  Isotope labeling of individual amino acids

2.  Nativity and functionality

DMPC/DMPA liposomes of Leptosphaeria rhodopsin expressed in P. pastoris (Fan et al, 2011, J. Biomol. NMR)

Preparing  your  spectrometer  Adamantane, 600MHz, Bruker TL2 probe, acquisition time of 400 ms, ~40 kHz decoupling Shimming

Magic angle adjustment using KBr or glycine

DMPC:DMPA=9:1, P:L 1:2.1 w/w PC:CL=8:2, P:L 1:2.1 w/w

15N spectra of Proteorhodopsin

Initial NMR sample screening

15N spectroscopy for initial screening 15N shift depends on the structure and environmental factors

Proteorhodopsin 800 MHz, 5 °C Expressed in E.coli

Leptosphaeria Rhodopsin (LR) 800 MHz, 5 °C P. Pastoris

ASR (2D crystals) 600 MHz, 5 °C E.coli

HAQP1 (2D crystals) 800 MHz, 5 °C Expressed in P. Pastoris

Is  crystallinity  important?  

Brown  and  Ladizhansky,  Protein  Science,  2015  

Two-­‐dimensional  NMR  :  an  example  of    13C-­‐13C  correla$on  spectroscopy  

a. -Hy b. C1x

c. C1xcosω1t1+C1ysinω1t1

d. C1zcosω1t1+C1ysinω1t1

e. (1-α)C1zcosω1t1+αC2zcosω1t1

f. (1-α)C1xcosω1t1+αC2xcosω1t1 Acq: (1-α)C1xcosω1t1exp(iω1t2) +

+ αC2xcosω1t1exp(iω2t2)

1st  scan   2nd  scan  a. -Hy b. -C1x

c. -C1xcosω1t1-C1ysinω1t1

d. -C1zcosω1t1-C1ysinω1t1

e. -(1-α)C1zcosω1t1-αC2zcosω1t1

f. -(1-α)C1xcosω1t1-αC2xcosω1t1 Acq: -(1-α)C1xcosω1t1exp(iω1t2) -

- αC2xcosω1t1exp(iω2t2)

ResulMng  signal  (subtracMon)    

(1-­‐α)C1xcosω1t1exp(iω1t2)  +            +αC2xcosω1t1exp(iω2t2)  

Two-­‐dimensional  NMR  :  an  example  of    13C-­‐13C  correla$on  spectroscopy  

a. -Hy b. C1x

c. C1xcosω1t1+C1ysinω1t1

d. C1xcosω1t1+C1zsinω1t1

e. (1-α)C1zsinω1t1+αC2zsinω1t1

f. (1-α)C1xsinω1t1+αC2xsinω1t1 Acq: (1-α)C1xsinω1t1exp(iω1t2) +

+ αC2xsinω1t1exp(iω2t2)

1st  scan   2nd  scan  a. -Hy b. -C1x

c. -C1xcosω1t1-C1ysinω1t1

d. -C1xcosω1t1-C1zsinω1t1

e. -(1-α)C1zsinω1t1-αC2zsinω1t1

f. -(1-α)C1xsinω1t1-αC2xsinω1t1 Acq: -(1-α)C1xsinω1t1exp(iω1t2) -

- αC2xsinω1t1exp(iω2t2)

ResulMng  signal  (subtracMon)    

(1-­‐α)C1xsinω1t1exp(iω1t2)  +            +αC2xsinω1t1exp(iω2t2)  

Two-­‐dimensional  NMR  :  an  example  of    13C-­‐13C  correla$on  spectroscopy  

ResulMng  signal  for  C1  

(1-­‐α)C1xcosω1t1exp(iω1t2)  +            +αC2xcosω1t1exp(iω2t2)  

ResulMng  signal  for  C2  

(1-­‐α)C2xcosω2t1exp(iω2t2)  +            +αC1xcosω2t1exp(iω1t2)  

Two-­‐dimensional  NMR  :  an  example  of    13C-­‐13C  correla$on  spectroscopy  

Combined  signal  for  C1  

(1-­‐α)C1xcosω1t1exp(iω1t2)  +            +αC2xcosω1t1exp(iω2t2)                                          &  

Combined  signal  for  C2  

(1-­‐α)C2xcosω2t1exp(iω2t2)  +            +αC1xcosω2t1exp(iω1t2)                                            &  

(1-­‐α)C1xsinω1t1exp(iω1t2)  +            +αC2xsinω1t1exp(iω2t2)  

(1-­‐α)C2xsinω2t1exp(iω2t2)  +            +αC1xsinω2t1exp(iω1t2)  

Real:      Imaginary:  

Two-­‐dimensional  NMR  :  an  example  of    13C-­‐13C  correla$on  spectroscopy  

Problem:  assume  that  tDARR  ~  T1.  

1.  Can  this  generate  arMfacts  in  the  2D  spectrum?    

2.  Modify  the  phase  cycling  to  eliminate  the  relaxing  component.    

Spectroscopic  Assignments  

Figure from: http://www.nmr.chem.uu.nl/~klaartje/STRUCT_BIOL/assignment/shiftc.gif

Carbon chemical shift distributions

13C chemical shift dependence: - AA residue type (~25 ppm)

-  Secondary structure type (5 ppm) -  Type of neighboring AA’s (2 ppm)

Knowledge of Cα gives Gly Cα AND Cβ gives A, T, S Cα AND Cβ AND Cγ gives most other AA His, Trp, Tyr, Phe are more difficult to identify because of the broader lines and/or lower side chain S/N

hYp://www.bmrb.wisc.edu/  :  general  staMsMcs  for  1H,  13C,  15N  shi\s  including  side  chains    

Useful resources for chemical shifts

Only  carbon  shi\s  shown  

2D  spectra  are  too  crowded  in  large  proteins  

Human  Aquaporin  1,  256  aa  

From 2D to 3D: Improving dispersion

Full side chains are detectable by 3D spectroscopy at affordable spectrometer time:

•  2D NCACX: 9hrs

•  3D NCACX: 3days

Shi et. al, BBA 2009, 1788: 2563

W,H,Y,I,F-­‐reversely  labeled  PR,  800  MHz  

Assignment Strategy

•  J-coupling based transfers

Sun et al, JACS 1997, 119, 8540; Hong, JBNMR 1999, 15, 1; Rienstra et al, JACS 2000, 122, 10979; Shi et al, JMB 2009, 386, 1078

•  Dipolar-based transfers •  Two-bond transfers shown by dashed arrows

       Solu$on  NMR                    i-­‐1                          i      

       Solid-­‐state  NMR                    i-­‐1                          i      

Assignment Strategy

Sun et al, JACS 1997, 119, 8540; Hong, JBNMR 1999, 15, 1; Rienstra et al, JACS 2000, 122, 10979; Shi et al, JMB 2009, 386, 1078

•  Short DARR mixing (20-50ms): Cα, Cβ •  Long DARR mixing (100-200ms): Cα, Cβ,Cγ, … •  Two-bond transfers shown by dashed arrows

       Extended  spin-­‐systems  CX(i-­‐1)-­‐CO(i-­‐1)-­‐N(i)-­‐CA(i)-­‐CX(i)  

Common  basic  element  of  heteronuclear  correla$on  spectroscopy:  NCA  &  NCO  

transfers  

PolarizaMon  can  be  directed  from  15N  towards  13CO  or  13CA  through  band-­‐selecMve    SPECIFIC  CP    

Selective transfers through SPECIFIC CP (NCO)

CO                                                                                CA  

ZQ  15N/13C  Hartmann-­‐Hahn  (HH)  Condi$on:     ωC ,eff −ω N ,eff = nω R , n = 1,2

ωC ,eff = ω1,C2 + Δω 2

ω N ,eff ~ω1N (on resonance)

Carrier frequency for NCO transfer (ΔωCO = 0)

ΔωCA ~ 120 ppm

Typical  CP  condi$ons  for  NCO  (800  MHz,  νR=15kHz):        

Choose ω1C ≈ωCO,eff ≈ 3.5νR = 52.5kHz

Choose ω1N ~ 2.5νR = 37.5kHz, HH is satisfied

ωCA,eff ≈ 52.5kHz2 + 24kHz2 ≈ 57.7kHz

ω1N ~ 2.5νR = 37.5kHz, HH is NOT satisfied

SPECIFIC  CP:  Baldus,  Petkova,  Herzfeld,  Griffin,  Molecular  Physics,  1998,  95:1197-­‐1207.    CP  tutorial:  Rovnyak,  Concepts  in  MagneMc  Resonance  A,  2008,  32A:  254-­‐276.    Advanced  theory  of  CP:  Marks  &  Vega,  J.  Magn.  Reson.,  1996,  118:  157-­‐172.  

x  

y  z  

ωCO,eff~ω1,C

x  

y  z  

ωCA,eff ΔωCA

ω1,C

Selective transfers through SPECIFIC CP (NCA)

CO                                                                                CA  

ZQ  15N/13C  Hartmann-­‐Hahn  (HH)  Condi$on:     ωC ,eff −ω N ,eff = nω R , n = 1,2

ωC ,eff = ω1,C2 + Δω 2

ω N ,eff ~ω1N (on resonance)

Carrier frequency for NCA transfer (ΔωCA = 0)

ΔωCO ~ 120 ppm

Typical  CP  condi$ons  for  NCA  (800  MHz,  νR=15kHz):        Choose ω1C ≈ωCA,eff ≈1.5νR = 22.5kHz

Choose ω1N ~ 2.5νR = 37.5kHz, HH is satisfied

ωCO,eff ≈ 22.5kHz2 + 24kHz2 ≈ 32.9kHz

ω1N ~ 2.5νR = 37.5kHz, HH is NOT satisfied

SPECIFIC  CP:  Baldus,  Petkova,  Herzfeld,  Griffin,  Molecular  Physics,  1998,  95:1197-­‐1207.    CP  tutorial:  Rovnyak,  Concepts  in  MagneMc  Resonance  A,  2008,  32A:  254-­‐276.    Advanced  theory  of  CP:  Marks  &  Vega,  J.  Magn.  Reson.,  1996,  118:  157-­‐172.  

x  

y  z  

ωCA,eff~ω1,C

x  

y  z  

ωCO,eff ΔωCO

ω1,C

Common  3D  SSNMR  experiments:    CANCO  

•  Typical  transfer  efficiencies:  NCO:  ~50%,  NCA:  35%  •  Of  the  three  experiments,  CANCO,  NCACX,  NCOCX,  CANCO  has  the  best  sensiMvity    

ASR (27kDa) @800 MHz Completely resolved

CA evolution

N evolution

CO evolution

For phases & setup procedures see Shi et al, Methods in Mol Biol. (2012) 895:153-65

Common  3D  SSNMR  experiments:    NCOCX/NCACX  

NCACX: DARR mixing of 20ms results in Cβ, Cγ, …

50ms results in Cβ, Cγ, Cδ…, as well as two-bond transfers (CA[i]èCO[i-1]) NCOCX: DARR mixing of 50ms results in Cα, Cβ

100ms results in Cα, Cβ, Cγ…, as well as two-bond transfers (CO[i-1]èCA[i])

N evolution

CA/CO evolution

CO,CA,CB… evolution

For phases & setup procedures see Shi et al, Methods in Mol Biol. (2012) 895:153-65

Long  aliphaMc  side  chains  can  be  detected,  e.g.  Leu,  Ile,  Val,  etc.      

Identification of residue type

Identification of residue type  

Shi, Ahmed, Zhang, Whited, Brown and Ladizhansky. J. Mol. Biol. (2009) 386, 1078–1093

Two-­‐  and  even  three-­‐bond  correlaMons  are  not  uncommon,  help  verify  assignments  

Observing “long-range” correlations

Building  Spin  Systems    

(i)  Peaks in the CONCA spectra are picked (ii)  N-CA shifts are matched with NCACX (iii) N-CO shifts are matched with NCOCX (iv) An extended spin system CX[i-i]-N[i]-CX[i] is

built

3D Sequential Assignments in PR

•  Spin Systems are linked by matching CO, C, C, Cg, etc. positions •  72 residues assigned in PR-FLY •  23 residues assigned in WHYFRI so far (data analysis in progress)

Spin system

Spin system

3D Sequential Assignments in PR

•  Spin Systems are linked by matching CO, C, C, Cg, etc. positions •  72 residues assigned in PR-FLY •  23 residues assigned in WHYFRI so far (data analysis in progress)

Spin system

Spin system

Cα@56.8

3D Sequential Assignments in PR

•  Spin Systems are linked by matching CO, C, C, Cg, etc. positions •  72 residues assigned in PR-FLY •  23 residues assigned in WHYFRI so far (data analysis in progress)

Spin system

Spin system

Spin System

Cα@56.8

3D Sequential Assignments in PR

•  Spin Systems are linked by matching CO, C, C, Cg, etc. positions •  72 residues assigned in PR-FLY •  23 residues assigned in WHYFRI so far (data analysis in progress)

Spin system

Spin system

Spin System

Spin System

Cα@56.8

3D Sequential Assignments in PR

•  Spin Systems are linked by matching CO, C, C, Cg, etc. positions •  72 residues assigned in PR-FLY •  23 residues assigned in WHYFRI so far (data analysis in progress)

Spin system

Spin system

Spin System

Spin System

Cα@56.8

3D Sequential Assignments in PR

•  Spin Systems are linked by matching CO, C, C, Cg, etc. positions •  72 residues assigned in PR-FLY •  23 residues assigned in WHYFRI so far (data analysis in progress)

Spin system

Spin system

Spin System

Spin System

Cα@56.8

 Another  example  of  a  backbone  walk  (ASR,  800  MHz)  

Reverse  labeling  

•   Spectral  simplificaMon    •   De  novo  assignments  are  more  complicated  because  of  interrupMons  in  backbone  walk  

Proteorhodopsin Proteorhodopsin Proteorhodopsin

SPC5  spectra  

Efficiency  of  reverse  labeling  

Proteorhodopsin  

Auxotrophic strains available to deal with scrambling: Lin, Sperling, Schmidt,Tang, Samoilova, Kumasaka, Iwasaki, Dikanov, Rienstra, Gennis, Methods  55  (2011)  370–378    

Reverse labeling simplifies backbone walk

L. Shi et al., Biochimica et Biophysica Acta 1788 (2009) 2563–2574

!

U-13C,15N PR W,HY,I,F-reversely labeled PR

13C  Spin  dilu$on  with  glycerol  

ASR,  229  residues,  800  MHz  

LeMaster, JACS, 1996, 118:9255-9264 Hong, JMR, 1999, 129, 389-401 Castellani et al, Nature 2002, 420:98-102.

13C labeling patterns using [1,3-13C] glycerol or [2-13C] glycerol as carbon sources

Sparse labeling helps assign aromatic residues

Black  –  U-­‐13C,15N  ASR;  Red      –  ASR  grown  on  2-­‐13C  glycerol        

Wang  et  al,  Biomol  NMR  Assign,  (2013)  7:253-­‐6  

13C  spin  diluMon  amplifies  side  chain  signals  of  aromaMc  residues,  enables  assignments  

Chemical Shift Index (CSI) plot for ASR

L. Shi et al., Angew. Chem. Int. Ed., 2011; S. Wang et al., Biomol. NMR Assign, 2013.

α-helix

β-strand

•  CSI=Secondary CS as a function of residue number •  Quick analysis of secondary structure, local distortions

TALOS torsion angle restraints

•  Backbone chemical shifts are sensitive to ψ, ϕ dihedral angles

Cornilescu, Delaglio, Bac, J. Biomol. NMR (1999), 13:289 Shen, Delaglio, Cornilescu, Bax, J. Biomol. NMR (2009) 44:213

Example  of  TALOS  predic$ons  in  ASR  

Wang et al, Nature Methods (2013), 10:1007.

Additional reading

L. Shi et al, J. Mol. Biol. (2009) 386:1078–1093; L. Shi et al., Biochimica et Biophysica Acta (2009) 1788:2563–2574; Sperling, Berthold, Sasser, Jeisy-Scott and Rienstra, J. Mol. Biol. (2010) 399:268–282; Higman, Flinders, Hiller, Jehle, Markovic, Fiedler, van Rossum, Oschkinat, J. Biomol. NMR (2009) 44:245–260 4D NMR 13C-detected NMR: Franks, Kloepper, Wylie, Rienstra, J. Biomol. NMR (2007) 39:107–131 L. Shi et al., Biochimica et Biophysica Acta (2009) 1788:2563–2574 Wylie, Bhate, and McDermott, Proc. Natl. Acad. Sci (2014) 111:185–190     Non-uniform sampling: Lecture by D. Rovnyak `

Structure determination

NMR-based Protein Structure Determination

Wüthrich, J. BioNMR, 2003, 27, 13.

Solution NMRNOE experiment

Solid State NMR proton-driven spin diffusion (PDSD)

Castellani et al, Nature 2002, 420, 98.

20 30 40 50 60

2D PDSD 13C-13C spectrum of U-13C,15N ASR

2D PDSD 13C-13C spectra of spin diluted ASR

1,3-13C Glycerol, 800 MHz, 500 ms PDSD mixing

2-13C Glycerol, 800 MHz, 500 ms PDSD mixing

Much  beYer  resoluMon  in  both  spectra,  many  (hundreds  to  thousands)  of  resolved  peaks  

Examples of 2D PDSD spectra of 2-ASR (500ms mixing, 800 MHz)

•  Many resolved peaks involving aromatic residues

Examples of 2D PDSD spectra of ASR (500ms mixing, 800 MHz)

•  Many resolved peaks involving aromatic residues •  Problem of ambiguous assignments (to be discussed later) •  Consistent patterns of cross peaks:

Y51Cα-Y11Cε2 & Y51Cγ-Y11Cα

D75Cα-W46Cδ2 & D75Cα-W46Cε3

A53Cα-H8Cγ & H8Ca-Y51Cγ

2D CHHC experiment on 1,3-ASR

Simplifying structure calculation (See lecture and tutorial by C. Schwieters)

1.  Introduce  intrahelical  H-­‐bonds  based  on  TALOS  and  Chemical  shi\  indexing      2.  Consider  only  long-­‐range  interhelical  peaks  by  using  symmetry  of  a  helix.    

-­‐  Group  I:  cross  peaks  that  can  be  explained  by  contacts  within|  i-­‐j|<5  

-­‐  Group  II:  cross  peaks  that  can  only  be  explained  by  contacts  within|  i-­‐j|>4  

-­‐  Group  II  represents  interhelical  contacts  

-­‐  Use  unambiguous  restraints  to  generate              de  novo  template  

Convergence  of  structure  calcula$on  

1-­‐2  days  

1-­‐2  days  

Wang et al, Nature Methods (2013), 10:1007.

   

Paramagnetic Relaxation Enhancements (PRE)

I.  Sengupta,  P.S.  Nadaud,  &  C.P.  Jaroniec,  Acc.  Chem.  Res,  2013      

AYach  paramagneMc  tag  to  a  protein:    Electron  spins  induce  large  PRE  effects  within  ~20  Å          AYenuaMon  of  cross  peak  intensiMes  is  distance  dependent    

Diamagne$c  reference     Paramagne$c  sample:  signal  akenua$on  within  ~20  Å  

Long-range distance restraints in GB1 (Nadaud, Helmus, Höfer, Jaroniec, J. Am. Chem. Soc. (2007), 129, 7502-7503)      

Blue cross-peaks - diamagnetic reference Red cross-peaks - paramagnetic sample

Cross  peak  decays  result  from    enhanced  transverse  relaxaMon    of  1H  and  13C  coherences  during  1H/15N  and  15N/13C  CP  

•  PRE  restraints  are  long-­‐range,  unambiguous!  

Lecture on paramagnetic NMR by B. Reif on Tuesday

Homonuclear  distance  measurements              

See also heteronuclear recoupling - lecture by C. Jaroniec

S2 ≈ − d122

d122 + d13

2 sin2 d122 + d13

2 t( )S3 ≈ − d13

2

d122 + d13

2 sin2 d122 + d13

2 t( )

Dipolar truncation in recoupling experiments

S2

S3

≈ d122

d132 = r13

r12

⎛⎝⎜

⎞⎠⎟

6

H DIPOLE ≈ H D13 + H D

12

H D12 << H D

13, H D12, H D

13⎡⎣ ⎤⎦ ≠ 0H DIPOLE

Effective ≈ H D13

The  effects  of  weak  couplings  are  removed  from  observable  dynamics!    

In homonuclear ZQ and DQ recoupling: Costa, PhD Thesis, 1996, MIT; Hohwy et al, J. Chem. Phys. (1999) 110, 7983. Bayro, et al., J. Chem. Phys. (2009) 130, 114506. In LGCP: Ladizhansky & Vega, J. Chem. Phys. (2000) 112, 7159.

Simulations of HORROR recoupling (ρ(0) = S1x )

Frequency-selective recoupling: Rotational Resonance (R2)

Spectra of 13C ZnAc as a function of mixing

0  ms  

1  ms  

3  ms  

5.5  ms  

7  ms  

D.P . Raleigh, M.H. Levitt, R.G. Griffin, Rotational Resonance in Solid State NMR. Chem. Phys. Lett., (1988), 146:71.

ΔCS = nν r , n = 1,2The R2 matching condition:

R2  Width  Measurement  

R2  

CW  45  kHz  

TPPM  79  kHz  

Signal =1

2[1− exp{−

ω (n) 2R2

ZQtmix

R2ZQ( )2

+ Δ2}]

P.R.  Costa  et  al.,  J.  Magn.  Reson.  (2003),  164,  92-­‐103;    Ramesh  et  al,  JACS  (2003),  125:15625;      Peng  et  al,  JACS,  (2008),  130,:359-­‐369      

Δ = δ1 − δ2 − nνrMeasures  deviaMon  from    the  R2  matching  condiMon  

Proton decoupling

Homogeneously Broadened Rotational Resonance (HBR2)

R. Janik et al., J. Magn. Reson., 2007; X. Peng et al., J. Am. Chem. Soc., 2008.

2D plane of a 3D experiment with DARR

2D plane of a 3D experiment with HBR2

22-­‐25  kHz  @600MHz  

Summary

•  13C-detected multidimensional spectroscopy is a versatile tool to study structure of immobilized proteins in the solid state

•  Not discussed in this presentation: - Fast and ultrafast MAS experiments - Multidimensional proton-detected NMR under ultrafast MAS and/or perdeuteration conditions

Students/postdocs  §  Shenlin  Wang  §  Sanaz  Emami  §  Meg  Ward    §  Lichi  Shi  §  Izuru  Kawamura  

       Collaborators  §  Leonid  Brown  (Guelph)  §  So-­‐Young  Kim  (Sogang  U)  §  Kevin  Jung  (Sogang  U)  §  Takashi  Okitsu  (Kobe  Pharm.  Univ.)  §  Akimori  Wada  (Kobe  Pharm.  Univ.)  

Leonid Shenlin Lichi Izuru Meg Sanaz

§  NSERC  §  Canada  Research  Chair  program  §  Canada  FoundaMon  for  InnovaMon  §  Ontario  Ministry  of  Research  and  InnovaMon