Mul$dimensional-correla$on- techniquesfor(membrane)protein … · 2016-01-10 · •...
Transcript of Mul$dimensional-correla$on- techniquesfor(membrane)protein … · 2016-01-10 · •...
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