lectures for chem 341 james aramini, phd

J.A.! 12/09/13!

Magnetic Resonance

Lectures for Chem 341

James Aramini, PhD.

CABM 014A [email protected]

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Outline""

1.  Introduction / Spectroscopy Overview!

2.  NMR Spectroscopy – Theory and Practice!

3.  Protein NMR & Resonance Assignment"

4.  NMR Structure Determination & !!Biological NMR Applications !

Dec. 4!!!!Dec. 9"!Dec. 11!

"

Lecture #2 Overview""

1.  NMR Experiments: From 1D to nD NMR!

2.  Protein NMR Spectroscopy!

3.  Resonance Assignment!!

Protein NMR Spectroscopy: A Short Course"

Sample and 1D 1H NMR Set-up:""I.  NMR tube in holder""II.  Sample in magnet and “lock”""III.  Tune desired nuclei"IV.   “Shim” "

"=> homogeneous field in coil region""V. Calibrate 90 deg. Pulse""VI. 1D 1H with presaturation:"“zgpr”"

relaxation!delay!

low power irradiation!of H2O signal (center)!

FID!

[ ]!ns! π/2 π

3π/2 2π


Aromatic

Imines Amides HCβ, γ, δ, ... HCα

wat

er

10 9 8 7 6 5 4 3 2 1 0

1H NMR of a real protein"NESG “ER541”: 125 a.a." ≈ 1 mM 15N-labeled; 600 MHz!

Chemical Shift (δ):" - intrinsic to chemical group! - very sensitive to surroundings "

very complicated!!!

DSS reference


From 1D to Multi-Dimensional NMR: Increase Resolution "

min

hrs

d

Time

ωo

t1

f2 (t2)

t1

A(t1)


Multi-Dimensional NMR: Increase Resolution "


Multi-Dimensional NMR: 2D 1H-1H NMR "

COSY""

TOCSY""

NOESY""

TOtal Correlation SpectroscopY""

COrrelation SpectroscopY""

NOE SpectroscopY""


Multi-Dimensional NMR: 2D 1H-1H NMR"

Ex. Valine " "• COSY"" " " "• TOCSY "

2D 1H-1H COSY""

2D 1H-1H TOCSY""


Multi-Dimensional NMR: Extending to Heteronuclei""Use Chemical Shift Dispersion of 13C and 15N"

HSQC: Heteronuclear Single Quantum Coherence "

2D 1H-15N HSQC"“Fingerprint of Protein”

Insensitive !Nuclei!Enhanced by!Polarization!Transfer!


Multi-Dimensional NMR: Extending to 3D""Use Chemical Shift Dispersion of 13C and 15N"

"Combine 2D “modules”: i.e., 3D 15N-edited TOCSY"

Hali!

HN!

N!

Ubiquitin"

HN!


Multi-dimensional NMR and Isotopic Labeling"

1 3 C 1 3 C

1 5 N 1 3 C

1 3 C 1 5 N

1 3 C 1 3 C

1 5 N

O

A A 3 O

A A 2 O

A A 1 H

H H

1H δ

15N δ

2D 15N-1H"

Isotope

Net Spin

γ / MHz T-1

Abundance / %

1H 1/2 42.58 99.98 15N 1/2 4.31 0.37 13C 1/2 10.71 1.108

15N δ

1H δ

1D 1H" 3D 1H-15N-1H"


Protein resonance assignment: Backbone "


Protein resonance assignment:""Example 3D Pulse Sequence: "" τ delays => J couplings "


Protein resonance assignment:"" "sequential (i-1) " " sequential + intra"

also: HN(CO)CACB also: HNCACB !!


Protein resonance assignment example: dvCcmE´""Cytochrome c maturation protein E"" ""

Aramini et al. (2012) Biochemistry 51, 3705!

Protein NMR Spectroscopy: A Short Course"Backbone resonance assignment: dvCcmE´…….Ala112"1H-15N HSQC" HNCO HN(CA)CO CBCA(CO)NH HNCACB "

HBHA(CO)NH HN(CO)CA HNCA"

HBi-1"

HAi-1"

Cí-1"Cí"Cí-1"

Cαi-1"

Cαi"

Cαi-1" Cα

i-1"

Cβi-1"

Cαi"

Cβi"

Cαi-1"

Cβi-1"

Protein NMR Spectroscopy: A Short Course"Backbone resonance assignment: connectivities"

Connect spin systems"with sequence"!i.e. HNCACB strips!

Kanelis et al. (2001) IUBMB Life 52, 291!

Protein NMR Spectroscopy: A Short Course"Backbone resonance assignment: dvCcmE´"Assigned 1H-15N HSQC"

Trp ε1!

sc NH2!

Arg ε (“aliased”)!

Protein NMR Spectroscopy: A Short Course"Backbone resonance assignment: dvCcmE´"“Connectivity Map” Moseley et al. (2001) Methods Enzymol 339, 91!

"

“3-rung matching”: C´, Cα, Cβ “CSI”: Chemical Shift Index!

Protein 2o Structure!Wishart & Sykes (1994) !J. Biomol. NMR 4, 171!

Protein NMR Spectroscopy: A Short Course"Backbone resonance assignment: Automated Assignment""Several Programs / Servers"!!!!!!!!!!!!!!!!!!!!!!Zimmerman et al. (1997) J. Mol. Biol. 269, 592!!

PINE!

Bahrami et al. (2001) PLoS Comput. Biol. 5, !e1000307!

EZ-ASSIGN!


Protein resonance assignment: Side Chain "" Assignment strategy for 13C/15N-labeled proteins"

(H)CC(CO)NH TOCSY"""""""""""""links side chain of i-1 to NH of i!

(H)CCH TOCSY / HCCH COSY""""""""""""" isolated side chain spin systems!


Protein resonance assignment: Side Chain "" Ex. Assignment of a Valine"

•  not stereoassigned ! !? Which Hγ bound to Cγ?!- Need HSQC, TOCSY and COSY !


Protein resonance assignment: Side Chain "" Ex. Assignment of a Valine"

C!

C!

C!

C!

* Methyls not stereoassigned!


Protein resonance assignment: Side Chain "" Stereospecific Leu/Val methyl assignments"

(S)"γ2/δ2"

(R)"γ1/δ1"

Fractional (10%) 13C labeling!

γ1!

δ1!γ2!

δ2!

Neri et al. (1989) Biochemistry 28, 7510!


Protein resonance assignment: Side Chain ""Stereospecific Leu/Val methyl assignments: i.e, dvCcmE´"

1H-13C HSQC (methyl region)"


Protein resonance assignment: Aromatic Side Chains "

Yamazaki et al. (1993) J. Am. Chem. Soc. 115, 11054!

3D CCH TOCSY !

3D (HB)CB(CG)CDHD 3D (HB)CB(CGCD)CEHE !


Protein resonance assignment: Aromatic Side Chains "

Pelton et al. (1993) Protein Sci 2, 543!

Protein NMR Resonance Assignment: 3D Expts"

1. Backbone " " " " 2. Side Chains"

" 3. NOESYs"

i - 1 i

HNCO!HN(CA)CO!CBCA(CO)NH!HNCACB!HBHA(CO)NH!HNCA!HN(CO)CA!! ≈ 3 days!

“through-!bond”!!

“through-space”!!

HCCH-TOCSY!CCH-TOCSY!HCCH-COSY!CC(CO)NH-TOCSY!

≈ 3 days!

≈ 4 days!15N-edited NOESY !13C-edited NOESYs!


Protein and Nucleic Acid nomenclature in NMR assignment:"IUPAC format"

" Amino Acids " " " Nucleotides"

Markley et al. (1998) J. Mol. Biol. 280, 944!


BioMagRes Data Bank (BMRB): "

http://www.bmrb.wisc.edu//nmr.html!


BioMagRes Data Bank (BMRB): "


8.22! 1.52!

1.62!

0.76!

0.73!

1.51!4.30!121.79!

55.70!

177.06!

42.26!24.70!

24.06!

26.78!

Leu"


BioMagRes Data Bank (BMRB): An example, 16272"


“bmrb” chemical shift text files!

PDB coordinates"2KIF"

(www.rcsb.org)"

Link to "JBC Paper"

PDB file"


Protein Bank (PDB): An example, 2KIF"

http://www.rcsb.org!Aramini et al. (201) J. Biol. Chem. 285, 13736!

NMR Spectroscopy: A Short Course"

Example Problem from Textbook:"0t

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lectures for chem 341 james aramini, phd

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