Mul$dimensional-correla$on- techniquesfor(membrane)protein … · 2016-01-10 · • Microcrystalline proteins/ •protein complexes • Amyloids • Membrane proteins • Cell walls,

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)


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)


ResulMng signal for C1


ResulMng signal for C2



Combined signal for C1

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

Combined signal for C2

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



Real: Imaginary:


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



Spin system

Spin system

Cα@56.8



Spin system

Spin system

Spin System

Cα@56.8



Spin system

Spin system

Spin System

Spin System

Cα@56.8



Spin system

Spin system

Spin System

Spin System

Cα@56.8



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

Documents

Mul$dimensional-correla$on- techniquesfor(membrane)protein … · 2016-01-10 · • Microcrystalline proteins/ •protein complexes • Amyloids • Membrane proteins • Cell walls,