Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Protein structure, dynamics and function by high-resolution NMR

Stephan Grzesiek, Aufbaustufe D4, FS 2011, 3.3.2011

[email protected]

What are the strong points of solution NMR of biomacromolecules?

 

works in solution

 

can determine (weak) macromolecular interactions

 

can determine dynamics from picoseconds to years

 

see protons

 

works with inhomogeneous systems (membrane protein suspensions, biofluids etc.)

 

works for unfolded proteins

 

can answer very specific questions by special spectroscopic methods (e.g. hydrogen bonds)

1

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Basics of protein structure and dynamics by solution NMR

Chemical Shifts in NMR Bo

ΔE = hω = hγB = hγB0 (1 − σ ) ω γ B B0 σ h

€

Larmor frequency gyromagnetic ratio = "magnetic moment" magnetic field at nucleus external magnetic field at nucleus0 shielding by electrons (chemical shift δ = -σ ) [ppm] Planck constant/(2π )

€

OH CH2

CH3

Staphylococcal nuclease

Ethyl alcohol ω

2

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



FID

Spectra

Fourier transform"

3

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



FT-NMR

free precession"

90˚ RF-Pulse"

I"

Free induction decay" "FID""

spectrum"

linewidth"

Fourier transform" t" exp(-t/T2)"

ω

Δω = 2/T2

Interactions between magnetic nuclei

4

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Basic 2D NMR

Transfer can be achieved by" 1.  NOE ("through space", NOESY) or" 2.  J-coupling ("through bond", COSY)"

2D 1H-15N COSY pulse sequence"

Also called HSQC (heteronuclear single quantum coherence)"

5

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



2D experiment after Fourier transform in second dimension: interferogram"

1H-15N

HSQC Cosy

protein tyrosine phosphatase 1B 298 aa ~ 35 kDa 1H 15N

15N

1H

ppm 6

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Example of 2D NOESY
 (ubiquitin)" 1H-1H

NOE"

"Through space"" ~ 1/r6"

More than two dimensions"

"mixn" :" always NOE " or J-correlation"

7

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Combination of 2D NOESY and 2D 1H-15N HSQC
 to 3D NOESY-15N-HSQC"

HSQC"

Slice of 3D NOESY-15N-HSQC"

2D NOESY"

8

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Protein backbone assign by combination of 3D triple resonance COSY experiments"

relevant J-couplings!

HNCO!

HNCA!

CBCA(CO)NH/HN(CO)CACB!

CBCANH/ HNCACB!

3D CT-HNCO, J. Magn. Reson. 96, 432-440 (1992)" HzNy → -Nx for improved 15N T2" =y"

O" H" C" N"

Hz" -Hy " HzNy → -Nx → -NyC'z" Nz C'y" ⇓" HxNz"

9

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Interferon-γ (31.4 kDa)" HNCA"

HNCO"

15N

= 118.9 ppm"

Ikura, Kay, Bax"

H" Hα" C

N

Cα" C

H"

O

N

Cα" C better:"

O

O

Hα"

HN(CO)CA"

10

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Connection sidechain to backbone: 3D CBCA(CO)NH"

S/N = e-3/6 e-6.6/8 e-7.4/14 e-22.4/52 e-22/42 e-4.5/13 • sin(2πJHβCβδ) • " • sin(2πJCαCβTAB) • cos(2πJCβCγTAB) • sin(2πJCαCβ ζ) " • sin(2π JCαCʻζ) • sin(2πJC'Nθ) • sin(2πJC'NTN) " • sin(2π JHNλ) ~ 0.09 - 0.17 * HNCO"

Hβ" Cβ"

O

Cα"

C"

Hα"

N" H"

CBCA(CO)NH of Interferon -γ"

11

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



NOE" 1H

r < ~ 5 Å"

1H

15N

15N 4D NOESY- 15N/15N HSQC"

δN =115.391 ppm δHN=8.758 ppm

δN =120.386 ppm δHN= 9.230 ppm

12

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



HIV-1 Nef"

Sample requirements

 

~ 0.25 ml 0.5 mM protein





(= 2.5 mg for 20 kDa protein)

  15N, 13C, (2H)

   

MWT MWT



labelled (E. coli)



< ~ 80 kDa for 3D structure



< ~100 (800) kDa for secondary

structure, functional tests, etc.

13

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR





Detection of molecular motions by NMR relaxation parameters

EN

ER GY

(radio frequency)"

nucleus

relaxation pathways"

 

after the nuclear spins in an NMR sample have been perturbed from their thermal equilibrium by RF pulses, they relax back to equilibrium with specific rates that depend on the motions of the spins."

 

therefore molecular motions can be detected by studying NMR relaxation phenomena"

Detection of ps to ns motions by NMR

Example: linewidth = 2/T2"

Overall motion! Cα" N"

H"

Fast motion" -> narrow linewidth"

Local motions"

C=O"

Slow motion" -> broad linewidth"

Macromolecule in solution"

NMR frequency"

14

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



T2 vs. T1 relaxation" Dephasing (loss of phase coherence) by transverse relaxation (T2): ~10-100 ms"

Remember: linewidth = 2/T2"

Return to (final) thermal equilibrium by longitudinal relaxation (T1): ~1-5 s"

The delay between experiments must be on the order of T1 to achieve return to equilibrium"

NMR relaxation rates

⎧ ⎫ 1/T1 ⎪ ⎪ ⎪ relaxation⎫ ⎪ ⎬ = ⎨1/T2 ⎬ ∝ Interaction 2 • J(transition frequency,correlation time) 1444444424444444 3 rates ⎭ ⎪ kNOE ⎪ spectral density function ⎪⎩ K ⎪⎭ RN (N z ) = 1/T1 = d 2 [ J(ω N − ω H ) + 3J(ω N ) + 6J(ω N + ω H )] + c 2 J(ω N ) RN (N x ) = 1/T2 = 0.5 d 2 [ 4J(0) + J(ω N − ω H ) + 3J(ω N ) + 6J(ω H ) + 6J(ω N + ω H )] +

€

c2 [4J(0) + 3J(ω N )] + Rex 6 γ H d2 NOE(H z ↔ N z ) = 1+ [6J(ω N + ω H ) − J(ω N − ω H )] γ N RN (N z )

€

d = strength of dipolar interaction" c = strength of chemical shift anisotropy" Rex = exchange contribution to line width"

15

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Detection of ps to ns motions by NMR

 

After the thermal equilibrium of the nuclear magnetic moments has been disturbed by the radio frequency pulses, the thermal motions of the molecules bring the nuclei back to the thermal equilibrium. This phenomenon is called nuclear spin relaxation.

 

Thus relaxation depends on the local and global motions of the molecule. In order to be effective for relaxation, the motions have to be in the time range of the magnetic transition frequencies, i.e. faster than Gigahertz. Hence the time window of ps to ns.

 

The finite linewidth of NMR resonances is one manifestation of relaxation.

 

In praxis, a number of relaxation parameters can be measured, such as T1 and T2 relaxation times and Nuclear Overhauser enhancements.

 

Evaluation of the data can give amplitudes and characteristic times of global and local motions.

16

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Detection of millisecond motions by NMR

 

NMR offers a second time range to detect motions within proteins. This time range is on the micro- to millisecond scale.

 

Motions in this time regime are observable from line broadening.

 

This line broadening is observed when groups of atoms move between different conformations at a frequency that is comparable to the difference in the NMR frequencies (chemical shifts) in the different conformations.

 

Typical differences of NMR frequencies in conformations are on the kHz scale. Hence the sensitivity to micro- to millisecond motions.

Detection of millisecond motions by NMR

Conformation A"

Conformation B"

H"

H" H"

H" C

exchange" time τ

C

|νA- νB| ~ kiloHertz"

νA"

NMR frequency"

νB" 17

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Detection of millisecond motions by NMR

Fast exchange"

Line" broadening"

Slow exchange"

NMR frequency"

Applications"  

Coupling of binding and folding in the c-di-GMP receptor PA4608 and in the transcription factor Brinker"

 

Conformational ensembles of unfolded proteins and protein folding"

18

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



cyclic-di-GMP causes bacteria to switch between two different life styles

[c-di-GMP]

[c-di-GMP]

planktonic"

sedentary"

motile single cells non-adhesive virulence factors acute virulence

sessile surface attached matrix embedded adhesion factors persistence

courtesy of U. Jenal

PA4608 is a c-di-GMP effector protein that blocks flagellar motor function

Apo PA4608! Ramelot, Arrowsmith, Kennedy! Proteins 2006!

PA4608 + c-di-GMP!

15N"

[ppm]"

1H"

19

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



PA4608 + c-di-GMP intermolecular contacts 103 intermolecular NOEs"

W77"

G1" 2 intermolecular h2JNNs"

G3" G2"

R13"

R9" 1H"

R8" 15N" 15N"

1H"

1H"

G2 H1"

G1 H1"

G3 H1"

G4 H1"

G4"

ppm"

PA4608 + c-di-GMP

RMSD: backbone protein core 0.2 Å, c-di-GMP bases 0.4 Å "

20

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



PA4608" N" C"

N"

C" Apo 


c-di-GMP complex"

Ramelot, Arrowsmith, Kennedy, Proteins 2006"

"

PA4608: exposure of surface charges upon c-di-GMP binding

21

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR

unfolded"



folded"

{1H}-15N NOE"

F. Cordier" M. Affolter"

Cordier et al., " JMB 2006, 361, 659"

22

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR





Increasing the capture radius by unfolding

The fly-casting mechanism" Shoemaker et al. PNAS, 2000(97), 8868"

Applications"  

Coupling of binding and folding in the c-di-GMP receptor PA4608 and in the transcription factor Brinker"

 

Conformational ensembles of unfolded proteins and protein folding"

23

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Foldon- The C-Terminal Domain From T4 Fibritin

Tao et al. (1997)

27 amino acids per monomer

Function: promote association

Structure of the Foldon Domain (3 x 27 aa) NMR:

1818 NOEs, 201 RDCs

Ramachandran core 92.1%

Backbone rmsd 0.31 Å

X-ray:

foldon in fibritin, Tao et al. (1997)

Meier et al. J Mol Biol, 2004. 344: 1051

24

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



160 µM Foldon: Trimer/Monomer transition " pH 4.2" Trimer"

pH 2.1" Trimer + Monomer"

Foldon: Trimer/Monomer transition "

15" 15"

5"

HSQC-Intensity"

δ13Cγ/δ"

5"

15" 5"

25

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



"A-state" = pH 2: Foldon monomer structure" isolated monomer"

monomer in trimer"

78 ROEs, 26 RDCs, 23 JHNHA"

Foldon monomer" β-hairpin Cα-shifts 
 ΔHf = -49.7 +/- 6.7 kJ/mol" ΔSf = -147 +/- 18 J/mol/K" Tm(all) = 334 +/- 9 K" Tm(D17) = 342 K"

26

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Tjandra + Bax, Science, 1997"

Residual orientation by mechanical pressure in acrylamide gel "

Sass et al. J Biomol. NMR 2000, 18, 303

27

Stephan Grzesiek, Aufbaustufe D4



squeeze

Protein structure, dynamics and function by high-resolution NMR



Protein G: observed RDCs to 1HN

Meier et al. J Am Chem Soc, 2003. 125: p. 44-5

28

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Protein G: structure from RDCs alone

RDCs only! (Meccano)! X-ray 1IGD

902 RDCs/56 residues" Phage + Otting phase alignment" Bouvignies et al. Angewandte Chemie, 2006, 45, 8166-9

backbone RMSD to X-ray: " 0.7 Å overall, 0.4 Å w/o first loop"



Protein G: structure from RDCs alone

RDCs only! (Meccano)

X-ray! 1IGD (1.1 Å)! 1PGA (2.1 Å)

RMSD β-strand 0.25 Å" Bouvignies et al. Angewandte Chemie, 2006, 45, 8166-9



29

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



∝ "

Foldon: Monomer RDCs in strained polyacrylamide gel "

1D

HN/Hz

Average 80˚C "

Meier et al. J Mol Biol (2004) 344,1051-69"

longitudinally" squeezed gel"

B0"

H

C

N

N

S

D NH = −

γ HγN r3

∫S

P (cosθ )dΩ ≥ 0 2

A. Annila"

30

€

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



trimer interface"

K16"

hairpin"

1D

HN/Hz"

Foldon Monomer: residual structure at 80˚C"

Average" 1.3 Hz"

D17"

P7" A6"

R8"

P4" E5"

Levinthal's paradox

E.g. folding of 100 amino acid protein:"   2 angles per amino acid"   only 3 possible values per angle"   1 ns to explore every conformation"

Folding would take 3200 * 1 ns = 2.7 1086 s " " " " "= 6.0 1068 * age of universe " " " " "[age of universe 14 Gyr (Hubble) = 4.4 1017 s]"

31

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Fitzkee, N. C., Fleming, P. J. and Rose, G. D. (2005)! Proteins, 58, 852-4.!

+ Sosnick, Blackledge= Staph. nuclease (Urea)!

Bernado et al. (2005) PNAS, 102, 17002! Jha et al. (2005) PNAS, 102, 13099!

Apomyoglobin (Urea)!

Coil library reproduces trends of unfolded state RDCs 32

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



RDCs in unfolded ubiquitin (urea, thermal) protonated sample"

deuterated sample"

long-range RDC correlations

1 H N -1 H N

1H N

Quantitative-J " COSY" 15N,

deuterated" ubiquitin" 8 M urea" pH 2.5, 25 ˚C" 10 % PAGE" ppm

33

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



RDCs in urea-unfolded ubiquitin Hα-Cα

coil model" (Blackledge, " Sosnick et al.)"

HN-N!

HNi- HNi+1!

coil model" biased " by 40 % to extended"

HNi- HNi+2! Hα i-1- HNi! RDC [Hz]"

residue"

Meier et al. J Am Chem Soc, 2007. 129: p. 9799-9807

long-range 1HN-1HN RDCs in β-turn (ubiquitin 8 M urea) 11/7"

10/7"

1H N

7/11" 7/10"

ppm

first β-turn" Meier et al. J Am Chem Soc, 2007. 129(4): p. 754-5

34

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



3J, CA, N, R2 sec. δ15N

sec. δ13Cα" [ppm]" 3J

HNHA"

isotropic" conditions" 15N

R2" [Hz]"

residue"

3J, CA, N, R2

ubiquitin"

× 4"

sec. δ15N

× 4"

sec. δ13Cα" [ppm]"

3J

HNHA"

15N

R2" [Hz]"

residue"

35

Stephan Grzesiek, Aufbaustufe D4



Protein structure, dynamics and function by high-resolution NMR

Direct observation of H-bonds" by J-couplings"

h3J

NC'"

Long-range HNCO (ubiquitin 8 M urea)

0.05 Hz"

0.06 Hz" 0.06 Hz"

0.05 Hz"

13C'"

[ppm]"

detected " H-bonds

long-range" RDCs

Meier et al. J Am Chem Soc (2007) 129, 754-755"

36

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Long-range information by paramagnetic relaxation enhancement (PRE)

+

MTSL"

ubiquitin single-cys mutants"

MTSL PREs of ubiquitin Cys-mutants 8M urea

ΔR2(15N) [s-1]"

K6C!

K48C!

S57C!

S20C!

prediction" for Gaussian" random chain"

K63C!

K35C!

G35C!

R74C!

Residue"

37

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Constrained ensemble structure calculation calculate" observables" RDC1, PRE1, …"

Conformer 1"

•" •" •"

average! calculated!

•" •" •"

, ! ,

!

observed!

refine"

…!

RDCobs, ! PREobs, ! …!

RDCn, PREn, …"

Conformer n"

Constrained ensemble structure calculation ubiquitin 8M urea PRE"

(s-1)"

RDC"

(s-1)"

Huang and Grzesiek. J Am Chem Soc (2010) vol. 132 pp. 694-705"

38

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Constrained ensemble structure calculation ubiquitin 8M urea RDC"

Huang and Grzesiek. J Am Chem Soc (2010) vol. 132 pp. 694-705"

Rg (Å)"

Normalized χ2"

RDC"

PRE"

SAXS value"

Cα-Cα contacts ubiquitin calculated unfolded ensembles" (400 x 10 conformers)" no constraints"

native state"

contact population"

all contacts"

native contacts"

RDC constr."

PRE constr."

RDC + PRE constr."

39

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Cα-Cα contacts ubiquitin 8M urea A-state"

"

RDC + PRE ensemble

native state"

all contacts"

native contacts"

putting in more data 626 RDCs (1DHN,1DHACA,1DCAC',DHNHA,DHNHN,1DCACB,1DHBCB,3DHAHB)

"

253 PREs" 354 3J (3JHNHA,3JC'HB ,3JNHB ,3JHAHB)" SAXS Gabel, Blackledge et al, JACS (2009), Schwieters & Clore Biochemistry (2007)

log(intensity)

SAXS

20 Conformers q [Å-1]

40

Stephan Grzesiek, Aufbaustufe D4

Protein structure, dynamics and function by high-resolution NMR



Cα-Cα contacts ubiquitin 8M urea all contacts"

1258 RDC, PRE, ! J, SAXS constraints!

population"

RDC, PRE, J, SAXS

native contacts"

!

RDC, PRE!

672 RDC, PRE! constraints!

radius of gyration [Å]"

PA4608! Martin Gentner" Urs Jenal" Tilman Schirmer "

Judith Habazettl"

Martin Allan"

RDCs in unfolded proteins! Sebastian Meier"

Navratna Vajpai, Martin Gentner, Martin Blackledge, IBS, Grenoble"

Unfolded protein ensemble calculations! Jie-rong Huang" Charles Schwieters, NIH"

SNSF, EU, " Boehringer Ingelheim, " Novartis, Roche"

41