from nmr spectra to structures - uzhand gln cγ < 35 ppm. residues with four carbons sidechain:...
TRANSCRIPT
![Page 1: From NMR spectra to structures - UZHand Gln Cγ < 35 ppm. Residues with four carbons sidechain: Pro and Arg can easily be distinguished by their Cδ shifts, for Pro Cδ ~ 50 ppm whereas](https://reader034.vdocument.in/reader034/viewer/2022042711/5f74739d6da7c4066c7baf45/html5/thumbnails/1.jpg)
• find conditions under which the protein does not aggregate is resonably stable
• measure nmr spectra
• sequence-specific sequential resonance assignment • identify spin systems • link spin systems (sequential assignment) • (stereospecific assignment of diasterotopic protons) • fully- interpret NOESY spectrum
• convert NOESY peak amplitudes into distances
• calculate 3D structure and refine the output
From NMR spectra to structures
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Chemical shifts (frequencies) and line intensities
11 10 9 8 7 6 5 4 3 2 1 0
backbone HN Hα
aliphatic
Me groups
aromatic
sidechain HN
ppm
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10 9 8 7 6 5 4 3 2 1 0 ppm
random coil amide
random coil methyl
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NOE information is used to introduce distance restraints into the structure
calculations
H
HNOE
γIγS(3cos2φ-1)r3
Bloc ~NOE ~ Bloc 2
“NOEs”
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From distances to 3D structures (II)
• Restrained molecular dynamics:
t
t+ΔtF
F = m ∂2r∂t2
F = ∂Upot∂r
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Upot = Ubond + Uangle + Udihedral + Uchiral + Uv.d.Waals
+ Ucoulomb + UNMR E p
ot
ddNOE
UNMR = UNOE + UJ + ….
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The origin of the NOE is dipolar (through-space) coupling of protons
R2,R1 ~ B2loc
γIγS(3cos2φ-1)
r3Bloc ~
BBoo
11HH
1515NN
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During the structure calculation only rotations about dihedrals are made
+180
120
-120
-120
60
60
-60
-60
0
0-180-180 120 +180
α−helix310-helix
α −helix(left-handed)
polyproline
parall. β−sheet
anti-parall. β−sheet
φ
ψ
Hi
NiC‘i-1
Ci-1α
Ci+1αNi+1
Hi+1
Oi-1
Ciβ
Ciα
C‘iOi
φi
ψi
ωi
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Güntert, Quarterly Reviews of Biophysics ��31 (1998), 145-237
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Automated calculation of NMR structures
Güntert, Quarterly Reviews of Biophysics ��31 (1998), 145-237
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Güntert, Quarterly Reviews of Biophysics ��31 (1998), 145-237
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Methods for assigning larger proteins
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N
CO
CαH
1H Frequencies
15N
Fre
quen
cies
15N,1H HSQC spectra are fingerprints of proteins
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14 6.56.66.76.86.97.07.17.27.37.47.5
110.0
110.5
111.0
111.5
112.0
112.5
*
*
*
*
*
*
15N spectra of proteins: Sidechain peaks
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Sequence-specific resonance assignment
in 13C,15N labelled proteins
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>>Use of 3-dimensional (tripleresonance) experiments
1H
15N
15N
1H
1H
1H
1H
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Backbone Assignment – 3D Experiments
HNCACB / HN(CO)CACB
HNCO / HN(CA)CO
HN(CACO)NH
15N NOESY
N Cα C N Cα C
Cβ
OH
H H
R
H H
CβH
O
R
H
H
N Cα C N Cα C
R
OHH H
R
OH
N Cα C N Cα C
R
OHH H
R
OH
N Cα C
H
R
OH
N Cα C N Cα C
Cβ
OH
H H
R
H H
CβH
O
R
H
H
N Cα C
H
CβH
O
R
H
H
residue i-1 residue i residue i+1
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Backbone Assignment – 3D Experiments
HNCACB / HN(CO)CACB
HNCO / HN(CA)CO
HN(CACO)NH
15N NOESY
N Cα C N Cα C
Cβ
OH
H H
R
H H
CβH
O
R
H
H
N Cα C N Cα C
R
OHH H
R
OH
N Cα C N Cα C
R
OHH H
R
OH
N Cα C
H
R
OH
N Cα C N Cα C
Cβ
OH
H H
R
H H
CβH
O
R
H
H
N Cα C
H
CβH
O
R
H
H
residue i-1 residue i residue i+1
N Cα C N Cα C
Cβ
OH
H H
R
H H
CβH
O
R
H
H
N Cα C N Cα C
R
OHH H
R
OH
N Cα C N Cα C
R
OHH H
R
OH
N Cα C
H
R
OH
N Cα C N Cα C
Cβ
OH
H H
R
H H
CβH
O
R
H
H
N Cα C
H
CβH
O
R
H
H
residue j-1 residue j residue j+1
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Backbone Assignment – 3D Experiments
HNCACB / HN(CO)CACB
HNCO / HN(CA)CO
HN(CACO)NH
15N NOESY
N Cα C N Cα C
Cβ
OH
H H
R
H H
CβH
O
R
H
H
N Cα C N Cα C
R
OHH H
R
OH
N Cα C N Cα C
R
OHH H
R
OH
N Cα C
H
R
OH
N Cα C N Cα C
Cβ
OH
H H
R
H H
CβH
O
R
H
H
N Cα C
H
CβH
O
R
H
H
residue i-1 residue i residue i+1
N Cα C N Cα C
Cβ
OH
H H
R
H H
CβH
O
R
H
H
N Cα C N Cα C
R
OHH H
R
OH
N Cα C N Cα C
R
OHH H
R
OH
N Cα C
H
R
OH
N Cα C N Cα C
Cβ
OH
H H
R
H H
CβH
O
R
H
H
N Cα C
H
CβH
O
R
H
H
residue j-1 residue j residue j+1
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sequential assignment strategy: building of fragments
1. picking of HN-C peaks -> spin systems (numbers 1-125) 2. alignment of 13C dimension for linking of correct successors/ predecessors
CA / COCA
CACB / COCACB
CG / COCG
NN
3. try to match fragments on amino acid sequence
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view of the 3D spectrum in CARA
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3D cube 15N 2D plane amide strip
Characteristic NH and N15 chemical shifts
HN
C13 N15
HN
2D 1H-15N HSQC is the root experiment of most of the standard triple-resonance (1H, 13C, 15N) NMR experiments used for backbone assignment. All the 3D triple resonance experiments are related by the common 1H,15N chemical shifts of the HSQC spectra: AMIDE STRIP
C13
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HNCACB / HN(CO)CACB
HNCO / HN(CA)CO
HN(CACO)NH
15N NOESY
Backbone Assignment – 3D Experiments
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http://www.bmrb.wisc.edu/ref_info/statful.htm
3 2 2 3 2 4 2 3 5 5 5 2 1 3 3 2 2 2 4 2
Standard Carbon Chemical shifts
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3 2 2 3 2 4 2 3 5 5 5 2 1 3 3 2 2 2 4 2
Standard Carbon Chemical shifts
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Can easily identify Gly, Ser/Thr and Ala from CB,CA shifts: Gly Cα ~45 ppm; Ser and Thr can be distinguished by Cβ shifts: Thr Cβ ~70 ppm ; Ser Cβ ~ 63 ppm. Ala Cβ chemical shifts is around ~18 ppm. Can group Leu, Tyr, Phe, Asn, Ile and Asp based on their Cβ shifts ~> 35 ppm. Differentiate between the residues having two (Asp, Asn, Trp, Tyr, Cys, His, Phe) carbons sidechains and those having 3 or more carbons in the sidechain by using CC(CO)NH. Among the residues having three carbon sidechain: Val, Met, Thr, Glu and Gln, Val has most upfield Cg chemical shifts. Ser/Thr can be distinguished Cg shift. Glu and Gln can be identified by their Cg shifts, Glu Cγ > 35 ppm and Gln Cγ < 35 ppm. Residues with four carbons sidechain: Pro and Arg can easily be distinguished by their Cδ shifts, for Pro Cδ ~ 50 ppm whereas for Arg Cδ ~43 ppm. Among the residues having five carbons side chain: Leu, Lys and Ile. Ile has the most upfield Cδ shifts ~10 ppm whereas Leu has Cβ ~ 43 ppm and Lys will have Cε ~43 ppm.
Identifying Residue type from chemical shifts
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3 2 2 3 2 4 2 3 5 5 5 2 1 3 3 2 2 2 4 2
Standard Proton Chemical shifts
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Side Chain Assignment Strategies
Side chain assignment:
N Cα C
Cβ
OHα
Hβ Hβ
H
CH HCH H
hCCH
CH
C
HC
H
HCcH
Identification of backbone protons:HBHA(CACBCO)NH HN(COCA)HA HACACO
N Cα C N Cα C
Cβ
OHα
Hβ H
R
H H
CβH
O
R
H
H
res i-1 res i
N Cα C
Cβ
OHα
Hβ H
R
HN Cα C N Cα C
Cβ
OHα
Hβ H
R
H H
CβH
O
R
H
H
res i-1 res i
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13C dimension strips of single spin systems for linking
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Prediction of secondary structure using chemical shifts using TALOS
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τc
1
0
-5
10-7 10-9 10-11
The magnitude of the 1H{15N}-NOE depends on the motional properties
Correlation time
15N {1H} -NOE
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NMR of metallothioneins
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Metallothioneins
� Small proteins: ~60 aa with ~30% cysteine residues� Coordinate metal ions
� No secondary structure elements� Two metal-thiolate clusters per protein:
� α-domain: 11 Cys coordinating 4 divalent metal ions� β-domain: 9 Cys coordinating 3 divalent metal ions
Crystal structure of MT-2 isolated from rat liver. (Romero-Isart N, Vasák M. J Inorg Biochem. Feb 2002)
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!
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N-term center C-term
center and C-term
Structures of Littorina littorea MT