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Institute of Biomedical SciencesAcademia Sinica
by NMR spectroscopyby NMR spectroscopy
Iren WangIren Wang王怡人王怡人
2008. May 8
III. Structure determination: Nucleic AcidsIII. Structure determination: Nucleic Acids
CBMB2008CBMB2008
Outline
I. Nucleic acids hold diverse structures and functionsI. Nucleic acids hold diverse structures and functions
a. In vitro SELEX a. In vitro SELEX
b. Diverse structures of nucleic acidsb. Diverse structures of nucleic acids
II. NMR Spectroscopy for Nucleic Acid AssignmentII. NMR Spectroscopy for Nucleic Acid Assignment
a. The building blocks of nucleic acidsa. The building blocks of nucleic acids
b. Resonance assignment in nucleic acidsb. Resonance assignment in nucleic acids
III. Some application cases for protein-nucleic acids complexes III. Some application cases for protein-nucleic acids complexes
IV. Others IV. Others (Advanced developments in NMR Spectroscopy)
a.a. Residual Dipolar Coupling (RDC) Residual Dipolar Coupling (RDC)
b. Transverse Relaxation-Optimized Spectroscopy (TROSY)b. Transverse Relaxation-Optimized Spectroscopy (TROSY)
c. Paramagnetic spin labelingc. Paramagnetic spin labeling
I. Nucleic acids hold diverse structures and functionsI. Nucleic acids hold diverse structures and functions
** ** the genetic information carriers
** ** tRNA: transporters of genetic information
mRNA: a copy of the information carried by a gene on the DNA
rRNA: a component of the ribosomes
snRNA (small nuclear RNA): important in a number of processes including RNA splicing and maintenance of the telomeres, or chromosome ends
** ** targets for proteins and/or drugs interaction
** ** the genetic information carriers
** ** tRNA: transporters of genetic information
mRNA: a copy of the information carried by a gene on the DNA
rRNA: a component of the ribosomes
snRNA (small nuclear RNA): important in a number of processes including RNA splicing and maintenance of the telomeres, or chromosome ends
** ** targets for proteins and/or drugs interaction
Nucleic acids hold diverse functionsNucleic acids hold diverse functions
Nucleic Acids Res. (2008) May, p.1-17
Postulated stem-loop diagram of the 5’ untranslated region of HIV-1HXB2 genomic RNA
Biochemistry (2008) 10, p.3283-93.
In vitro SELEX In vitro SELEX (systematic evolution of ligands by exponential enrichment) (systematic evolution of ligands by exponential enrichment)
an excellent tool for finding nucleotide molecules that have a high affinity for a particular target from a random pool under specific conditions. Three processes: Selection of ligand sequences, Partitioning of aptamers, amplification of bound aptamers
Anal Bioanal Chem (2007) 387, p.171-82.
Nucleic acids hold diverse structuresNucleic acids hold diverse structures
DNA duplexes, triplex, multi-stranded G-quadruplex structures
RNA structural elements: helices, hairpins, bulges, junctions, pseudoknots
** non-helical conformations and tertiary structure are stabilized by: metal ions, water-mediated H-bonds, and stacking interactions
** formation of a double-stranded helix is driven by cooperative attractive hydrogen bonding and stacking interactions
** U-turn / reversed U-turn of RNA: sharp turns in hairpin loops
** the diversity of RNA structures compared to DNA is a result of non-helical secondary structure
** non Watson-Crick base pairs are important for RNA/RNA and RNA/protein recognition
The possible conformations formed by poly-nucleotides in solution are flexible, “unstructured” single strands, stacked helical single strands, hairpins, regular duplexes formed by
complementary strands, and a variety of aggregates between partially complementary strands, which may contain bulges, dangling ends, or stacked single stranded ends.
The arrangement of hydrogen bonds between
guanines in a G-tetrad
Telomeric DNA quadruplex structures Telomeric DNA quadruplex structures
Current Opinion in Structural Biology (2003) 13, p.275-83.
Stem 2
Loop 1Loop 1
Secondary structure of the T arm and pseudoknotted acceptor arm Secondary structure of the T arm and pseudoknotted acceptor arm of the tRNA-like structure of TYMV genomic RNAof the tRNA-like structure of TYMV genomic RNA
Science (1998) 280, P.434-8.
RNA structureRNA structure
By Michael Sattler
Proteins recognize unusual RNA structural elementsProteins recognize unusual RNA structural elements
Structure (2000) 8, R47-R54.
RNA structure: protein recognition
By stabilizing an adjacent interaction surface, bulges can participate in complex protein binding sites.
stacked flipped-out
groove-binding flap residues
Structure (2000) 8, R47-R54.
RNA bugles as architectural and recognition motifsRNA bugles as architectural and recognition motifs
Bulges: unpaired stretches of nucleotides located within one strand of a nucleic acid duplex -sizes: vary from a single unpaired residue up to several nucleotides
Bugles stabilization by metal ions Bugles distortions
Structure (2000) 8, R47-R54.
RNA bugles as architectural and recognition motifsRNA bugles as architectural and recognition motifs
Non-Watson–Crick base pairs employ non-standard H-bondsNon-Watson–Crick base pairs employ non-standard H-bonds
Structure (2000) 8, R55-R65.
bifurcated H-bonds
C-H N/O H-bond
water-mediated
cis Watson-Crick G*A
open, water-mediatedWatson-Crick G*A
II. NMR Spectroscopy for Nucleic Acid AssignmentII. NMR Spectroscopy for Nucleic Acid Assignment
Nucleic acid bases, nucleosides, nucleotidesNucleic acid bases, nucleosides, nucleotides
Nomenclature, structures, and atom numbering for the sugars contained in
common Nucleotides.
Nomenclature, structures, and atom numbering for the basesbases contained in common Nucleotides.
Labile protons
Torsion Angles in Nucleic AcidsTorsion Angles in Nucleic Acids
By Michael Sattler
Sugar pucker, pseudorotationSugar pucker, pseudorotation
A. Puckering of five-membered ring into envelope (E) and twist (T) forms.B. Definition of sugar puckering modesC. Pseudorotation cycle of the furanose ring in nucleosides.
(A) (B) (C)
NMR of Proteins and Nucleic acids (1986) by Kurt Wüthrich
Syn/anti conformations – the χ torsion angleSyn/anti conformations – the χ torsion angle
NMR of Proteins and Nucleic acids (1986) by Kurt Wüthrich
1D 1D 11H NMR spectrum in Nucleic Acids (in DH NMR spectrum in Nucleic Acids (in D22O)O)
H2 H6 H8 H1’ H5 H2’ H3’ H4’ H5’ H5’’ CH3
imino amino aromatic
1D 1D 11H NMR spectrum in Nucleic Acids (in HH NMR spectrum in Nucleic Acids (in H22O)O)
Flowcharts for resonance assignment in nucleic acidsFlowcharts for resonance assignment in nucleic acids
A. NOE-based assignment in unlabeled nucleic acidsA. NOE-based assignment in unlabeled nucleic acidsA. NOE-based assignment in unlabeled nucleic acidsA. NOE-based assignment in unlabeled nucleic acids
Progress in Nuclear Magnetic Resonance Spectroscopy (1998) 32, p.287-387.
I (H2O) Assignment of imino (and amino) resonances to establish base pairing NOESY imino-imino, amino-imino
II (H2O) Partial resonance assignment of non-exchangeable protons via NOE connectivities to amino and/or imino protons NOESY imino-H2/H6/H8/H5/H1’
III (D2O) Identification of sugar proton spin systems (mainly H1’/H2’/H2’’/H3’) (1H, 1H) COSY/TOCSY
Identification of aromatic spin systems (Cytosine/Thymine H5/H6) (1H, 1H) COSY/TOCSY
Sequential resonance assignment NOESY H6/H8-H1’, H6/H8-H2’H2’’
IV (D2O) Assignment of 31P resonances and confirm/extend H3’,H4’,H5’,H5’’ assignments (1H, 31P) HETCOR/HETTOC
Flowcharts for resonance assignment in nucleic acidsFlowcharts for resonance assignment in nucleic acids
B. NOE-based assignment in labeled nucleic acidsB. NOE-based assignment in labeled nucleic acidsB. NOE-based assignment in labeled nucleic acidsB. NOE-based assignment in labeled nucleic acids
I (H2O)
Exchangeable proton/nitrogen correlation 2D 15N-HMQC imino 1H optimized G N1H, U N3H amino 1H optimized C N4H2, G N2H2, A N6H2
Exchangeable proton/nitrogen sequential assignment 3D 15N-NOESY-HMQC (imino 15N edited NOESY) imino-imino, amino-imino 3D 15N-NOESY-HMQC (amino 15N edited NOESY) amino-imino
II (H2O) Partial resonance assignment of non-exchangeable proton from NOE connectivities with amino and/or imino protons 3D 15N-NOESY-HMQC (imino 15N edited NOESY) aromatic-imino
Progress in Nuclear Magnetic Resonance Spectroscopy (1998) 32, p.287-387.
Flowcharts for resonance assignment in nucleic acidsFlowcharts for resonance assignment in nucleic acids
III (D2O) Identification of sugar proton spin systems 3D HCCH-COSY H1’-H2’ 3D HCCH-RELAY H1’-H2’/H3’ 3D HCCH-TOCSY
Identification of sugar carbon spin systems 2D 13C-CT-HSQC/HMQC 3D HCCH-COSY H1’-C2’ 3D HCCH-RELAY H1’-C2’/C3’ 3D HCCH-TOCSY H1’-C2’/C3’/C4’/C5’
Identification of proton/carbon aromatic spin systems 2D 13C-CT-HSQC/HMQC H6-C6, H8-C8, H5-C5, H2-C2 2D/3D HCCH-COSY H6-H5, H6-C6/ C5, H5-C6/ C5
Sequential resonance assignment 3D 13C-NOESY-HMQC H6/H8-H1’, H6/H8-H2’H2’’
IV (D2O) Assignment of 31P resonances e.g. (1H, 31P) HETCOR/HETTOC
B. NOE-based assignment in labeled nucleic acidsB. NOE-based assignment in labeled nucleic acidsB. NOE-based assignment in labeled nucleic acidsB. NOE-based assignment in labeled nucleic acids (continued)
Progress in Nuclear Magnetic Resonance Spectroscopy (1998) 32, p.287-387.
C. Assignment via through-bond coherence transfer in labeled nucleic acidsC. Assignment via through-bond coherence transfer in labeled nucleic acidsC. Assignment via through-bond coherence transfer in labeled nucleic acidsC. Assignment via through-bond coherence transfer in labeled nucleic acids
Flowcharts for resonance assignment in nucleic acidsFlowcharts for resonance assignment in nucleic acids
Progress in Nuclear Magnetic Resonance Spectroscopy (1998) 32, p.287-387.
I (H2O) Exchangeable proton/nitrogen correlation 2D 15N-HMQC imino 1H optimized G N1H, U N3H amino 1H optimized C N4H2, G N2H2, A N6H2
II (H2O) Through-bond amino/imino to non-exchangeable base proton correlations HNCCH/HCCNH
III (D2O) 1. Through-bond H2-H8 correlations {HCCH-TOCSY/(1H,13C) HMBC}
2. Through-bond base-sugar correlations {HCN (base) with HCN (sugar), HCNCH, HCNH, {HCN (sugar) with H8N9(H8)C8H8}, {HCN (sugar) with (Hb,Hb) HSQC}, {(H1’, C8/6) HSQC with (H8/6, C8/6) HSQC}
3. Through-bond sugar correlations {HCCH-COSY/ HCCH-TOCSY}
4. Sequential resonance assignment via through-bond sugar-phosphate backbone correlations (1H, 13C, 31P) HCP/ PCH/ PCCH-TOCSY/ HPHCH
NOE-based assignment in NOE-based assignment in unlabeled nucleic acidsunlabeled nucleic acids
Imino Proton Assignments by 2D NOESY spectrum
2G
18T
17G
5G
15T
7T
8G
12T
11G
a
b
imino proton
imin
o p
roto
n
5'–CGACGATGACGTCATCGTCG-3' 3'-GCTGCTACTGCAGTAGCAGC-5'
1 5 10 15 20
20 15 10 5 1
I. Assignment of imino (and amino) resonances in H2O
N
N
O
N
R
H
H
H
HN
N
N
N
N
O
R
H
H
H
H
G C
N
N
OR
OMe
H N
N
N
N
R
N
H
H
H
H
H AT
J. Chin. Chem. Soc. (1999) 46, p.699-708.
Imino Proton to Amino to Aromatic ProtonsImino Proton to Amino to Aromatic Protons
II. NOESY imino-H2/H6/H8/H5/H1’ in H2O
b. Imino to amino/aromatic
c. Amino to aromatic
d. Aromatic to H2’/H2”
N
N
O
N
R
H
H
H5
H6N
N
N
N
N
O
R
H
H
H8
H
G C
a. Imino to imino
2
3
4
N
N
OR
OMe
H6 N
N
N
N
R
N
H2
H
H
H8
H AT
1
6
5 7
Imino Proton to Amino and AromaticImino Proton to Amino and Aromatic
II. NOESY imino-H2/H6/H8/H5/H1’ in H2O
N
N
O
N
R
H
H
H5
H6N
N
N
N
N
O
R
H
H
H8
H
G C
N
N
OR
OMe
6H N
N
N
N
R
N
H2
H
H
H8
H AT
H5
H2
b. Imino to amino/aromatic
J. Chin. Chem. Soc. (1999) 46, p.699-708.
Only intra-strand aromatic to aromatic connectivitiesOnly intra-strand aromatic to aromatic connectivities
III. Identification of aromatic spin systems in D2O
J. Chin. Chem. Soc. (1999) 46, p.699-708.
III. Sequential resonance assignment in D2O
NOESY H6/H8-H1’, H6/H8-H2’H2’’NOESY H6/H8-H1’, H6/H8-H2’H2’’
Cytosine: CH5-CH6
JMB (1983) 171, p.319-36.
Duplex-hairpin 5'–CGCGTATACGCG-3'
Nucleic Acids Res. (1985) 13, p.3755-72.
Only intra-residue cross peaks were marked. a-f. are the six big CH5-CH6 cross peaks.
III. Sequential resonance assignment in D2O
NOESY H6/H8-H1’NOESY H6/H8-H1’
J. Chin. Chem. Soc. (1999) 46, p.699-708.
Only intra-residue cross peaks were marked.
III. Sequential resonance assignment in D2O
NOESY H6/H8-H2’H2’’NOESY H6/H8-H2’H2’’
J. Chin. Chem. Soc. (1999) 46, p.699-708.
5’ G-p-C-p-G-p-A-p-T-p-A-p-G-p-A-p-G-p-C-p-G G-p-C-p-G-p-A-p-G-p-A-p-T-p-A-p-G-p-C-p-G 5’
2 3 4 5 6 7 8 9 10 11
11 10 9 8 7 6 5 4 3 2
(n-1) H3'- (n) P (n) P - (n) H4'
11H-H-3131P Correlation SpectrumP Correlation Spectrum
7p3p
6p
2p, 5p, 9p
11p
10p
8p4p
7G
3G
8A
4A
10C
11G5T
2C9G
6A
7G3G
6A2C
5T
8A
1G4A
10C9G
Phosphate buffer
b) 3
1P
OH5'
H5"
H4'
O
Base
H1'
H2"
H2'
5'
H4'
O
P
H3'
O
O
(t)
O
Base
H1'
H2"
H2'
3'
H3'
-
JACS (1992) 114, 3114-5.
NOE-based and via through-bond coherence transfer NOE-based and via through-bond coherence transfer assignment in assignment in labeled nucleic acidslabeled nucleic acids
RNA synthesis by in vitro transcription RNA synthesis by in vitro transcription
dT rA
dC rG
dG rC
dA rU
Transcription
DNA
RNA
3’ ATTATGCTGAGTGATATCCTTATACTATGTAAACTAGTCATATAGG 5’
5’ TAATACGACTCACTATAG 3’
DNA template
Top strand
Transcription startingT7 polymerase
RNA synthesized GGAUUAUGAUACAUUUGAUCAGUAUAUCC5’ 3’
RNA samples at natural isotopic abundance and enriched in 15N and 13C can be prepared with T7 RNA polymerase.
Heteronuclear Chemical Shifts in NucleotidesHeteronuclear Chemical Shifts in Nucleotides
Current Protocols in Nucleic Acid Chemistry (2000) 7.7.1-7.7.30
2D 2D 1515N–N–11H HMQC spectra of RNA imino rH HMQC spectra of RNA imino resonances at different conditions esonances at different conditions
PNAS (1997) 94, p.2139-44.
The The 11H-H-1313C HSQC spectra of labeled nucleic acidsC HSQC spectra of labeled nucleic acids (A) H6/H8-C6/C8, (B) H1’-C1’, (C) H2’/H2’’-C2’, and (D) H3’-C3’ (A) H6/H8-C6/C8, (B) H1’-C1’, (C) H2’/H2’’-C2’, and (D) H3’-C3’
Adenine
H8-H1’ correlation, HCNHCN
H1’,H2’, H3’ H4’H5’H5” correlations, HCCH-TOCSYHCCH-TOCSY
Nucleotide spin system
H2-H8 or H5-H6 correlation
Intraresidue correlation Intraresidue correlation
via through-bond coherence transfer NMR experimentsvia through-bond coherence transfer NMR experiments
Correlation in the base-sugar: HCN 3D spectraCorrelation in the base-sugar: HCN 3D spectra
2D and 3D TROSY-HCN for obtaining ribose base and intra-base 2D and 3D TROSY-HCN for obtaining ribose base and intra-base correlations in the nucleotides of DNA and RNA. correlations in the nucleotides of DNA and RNA.
Dotted arrows indicate the intra-base transfers and solid arrows the ribose-base transfers.
JACS. 2001, 123, 658-64.
H6/H8 H1’
3D HCCH-TOCSY3D HCCH-TOCSY
Progress in Nuclear Magnetic Resonance Spectroscopy (1998) 32, p.287-387
H1’
Interresidue correlation through bond (HInterresidue correlation through bond (HCCPP))
Adenine
OH
GuanineH3’-C3’-P(n-1)
Residue n
H5’,H5’’(n-1)-C5’(n-1)-P(n-1)
2 spin systems can be linked
Progress in Nuclear Magnetic Resonance Spectroscopy (1998) 32, p. 287-387
Direct observation of H-bonds in nucleic acid base pairs Direct observation of H-bonds in nucleic acid base pairs by inter-nucleotide by inter-nucleotide 22JJNNNN couplings couplings
JNN HNN-COSY JHN HSQC 3D13C-NOESY
1H3-15N3(U) to 15N1(A)
1H2 (A) to 15N1(A) and 15N3(A) JACS. (1998) 120, 8293-7.
the connectivity between 1H3(U329) and the 1H2-13C2 (A32)
Structural Determination of Nucleic Acids by NMRStructural Determination of Nucleic Acids by NMR
1)1) Similar to those used in proteinSimilar to those used in protein
2)2) First build a nucleic acid First build a nucleic acid sequence templatesequence template
3)3) Input Input H-bonded constraintsH-bonded constraints
4)4) Input all exchangeable and non-exchangeable Input all exchangeable and non-exchangeable distance constraints distance constraints and/orand/or dihedral dihedral constraintsconstraints
5)5) Use Use Distance GeometryDistance Geometry calculation to get some initial structures calculation to get some initial structures
6)6) Use Use Molecular DynamicsMolecular Dynamics method to refine the structures method to refine the structures
Distance constraints (NOEs)Dihedral angles constraints (J-coupling)
Distance geometry calculator(XPLOR-NIH)
5’
3’
5’
3’
III. Some application cases for protein-nucleic acids III. Some application cases for protein-nucleic acids complexescomplexes
NMR spectroscopy as a tool for secondary structure dNMR spectroscopy as a tool for secondary structure determination of large RNAs.etermination of large RNAs.
Annu. Rev. Biophys. Biomol. Struct. (2006) 35,p.319-42.
Annu. Rev. Biophys. Biomol. Struct. (2006) 35,p.319-42.
The structure of HCV IRES domain IIThe structure of HCV IRES domain II
Dependence of RDC values on the orientation of the interdipolar vector (C-H) and the alignment tensor
Rmsd = 7.48Å Rmsd = 5.79Å Rmsd = 2.18Å
Refinement of the HCV IRES domain II structures calculated by the use of different sets of RDCs
The common DNA recognition motifsThe common DNA recognition motifs
By Dr. Song Tan
Helix-turn-helix (HTH) domainHelix-turn-helix (HTH) domain Zinc-finger (ZF) domainZinc-finger (ZF) domain
The common DNA recognition motifsThe common DNA recognition motifs
Winged-helix (WH) domainWinged-helix (WH) domain
Chemical shift change plot based on NMR titration data
E172 N196 S204 G213
N196
C-N-
S204
G213
E172
33
22
11
Wing
Recognition helix
Structure (1997) 5, p.559-70.
RNP/RRM domainRNP/RRM domain
The common RNA recognition motifsThe common RNA recognition motifsRNP: ribonucleoproteinRRM: RNA-recognition motif
The KH domainsThe KH domains
Structure (1999) 7, p.191-203.
The common RNA recognition motifsThe common RNA recognition motifs
KH: K-homology motif
Science (1998) 279, p. 384-8.
Structure of the HIV-1 Nucleocapsid protein with SL3 Structure of the HIV-1 Nucleocapsid protein with SL3 -RNA recognition element-RNA recognition element
Science (1998) 279, p. 384-8.
Structure of the HIV-1 Nucleocapsid protein with SL3 Structure of the HIV-1 Nucleocapsid protein with SL3 -RNA recognition element-RNA recognition element
Science (1998) 279, p. 384-8.
The best fit superposition and space-filling representation The best fit superposition and space-filling representation of the SL3 RNA in the NC-SL3 complexof the SL3 RNA in the NC-SL3 complex
Recognition of the mRNA AU-rich element by the Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11dzinc finger domain of TIS11d
NSMB (2004) 11, p. 257-64.
(a) The ensemble of best 20 structures superposed on backbone heavy atoms in ordered regions of the protein and RNA. (b) Ribbon representation of a single structure with the addition of green side chains for the zinc-coordinating ligands. (c) Backbone superposition of the structure ensembles of fingers 1 and 2. Finger 1 (Arg153–Phe180) is dark blue (backbone), green (zinc coordinating side chains) and red (intercalating aromatic rings); the bound RNA (U6, A7, U8, U9) is orange. The corresponding colors for finger 2 are light blue, yellow, pink and yellow.
EMBO J (2000) 19, p.6870-81.
Comparison between nucleolin RBD12-sNRE complex aComparison between nucleolin RBD12-sNRE complex and the other RBD-RNA complexesnd the other RBD-RNA complexes
RBD: RNA biding domain
IV. Advanced developments in NMR SpectroscopyIV. Advanced developments in NMR Spectroscopy
New Techniques in NMR SpectroscopyNew Techniques in NMR Spectroscopy
(1). Residual Dipolar Coupling (RDC)
(2). Transverse Relaxation-Optimized Spectroscopy (TROSY)
(3). Other Applications
NOE, dihedral angle and H-bond are short-range restraints and have limitations for some structure determination, like extended structures or multiple-domain structure. RDC is a novel restraint and provides global structure information.
or
TROSY, which was developed by K. Wüthrich, can select one fourth of the signals that relax more slowly than the others. The utilization of TROSY techniques push the size limit of NMR spectroscopy to 30~50 kDa.
Paramagnetic Spin Labeling.
Residual Dipolar Coupling (RDC)Residual Dipolar Coupling (RDC)
Residual dipolar couplings arise from dipole-dipole interactions between nuclei. In aqueous solution, the isotropic orientation of the molecules average out the dipolar couplings. However, in oriented media, the molecular tumbled anisotropically. The order of 10-4 to 10-3 of anisotropy tuned the dipolar coupling constant to be a residual value of few Hz, which are well detectable by NMR spectroscopy.
Values of static dipolar coupling constant of two-spin systems in protein backbone
The residual dipolar coupling between two spins A and B are given by :
<DAB> = - C(Bo) [ a(3cos2 -1) + 3/2 r(sin2 cos2) ]
where
C(Bo) = S(Bo2/15kT)[AB h/(42rAB
3).
A and B are gyromagnetic ratios of A and B.
rAB is the distance between A and B.
Residual Dipolar Coupling (RDC)Residual Dipolar Coupling (RDC)
So, Bo , DAB
S (order parameter) , DAB
• Phages (Pf1, fd, TMV) (Zweckstetter, JBNMR)
• Bicelles (Sanders & Schwonek, Biochemistry, 1992; Ottiger&Bax, JBNMR 1998)
• Polyacrylamide gels (Tycko,JBNMR; Grzesiek JBNMR; Chou, JBNMR)
• Paramagnetic tagging (Opella , Griesinger, Byrd)
• CPBr/hexanol (Barrientos, J. Mag. Res, ~2000)
• C12E5/hexanol (Ruckert&Otting, JACS 2000)
• Cellulose crystallites (Matthews, JACS, ~2000)
Alignment Media
The most-used media for RDC measurement are :
(a). Phospholipid bicelles and (b). Filamentous phage
Alignment of Molecules in Anisotropic SolutionsAlignment of Molecules in Anisotropic Solutions
1H Chemical Shift (ppm)
Isotropic solution + 5.3 mg/ml Pf1
15 N
Ch
em
ical
Sh
ift
(pp
m)
15N-IPAP HSQC for HN RDC values
JNH JNH + DNH
1H Chemical Shift (ppm)
Isotropic solution + 5.3 mg/ml Pf1
13 C
Ch
em
ical
Sh
ift
(pp
m)
3D HNCO for C’N RDC values
JC’N
JC’N + DC’N
Structure Refinement with RDC RestraintsStructure Refinement with RDC Restraints
X
Y
Z
<DAB>() = Da [ a(3cos2 -1) + 3/2 Dr(sin2 cos2) ]
1. Main relaxation source for 1H and 15N: dipole-dipole (DD) coupling and, at high magnetic fields, chemical shift anisotropy (CSA).
2. Different relaxation rates (line width) for each of the four components of 15N-1H correlation.
3. The narrowest peak (the blue peak) is due to the constructive canceling of transverse relaxation caused by chemical shift anisotropy (CSA) and by dipole-dipole coupling at high magnetic field.
4. TROSY selectively detect only the narrowest component (1 out of 4).
15N
1H
(a). None-decoupled HSQC (b). Decoupled HSQC (c). TROSY-HSQC
Transverse Relaxation-Optimized Spectroscopy (TROSY)
DD + CSA(large) (large)
DD – CSA(large) (large)
DD + CSA
(large) (small)
DD – CSA
(large) (small)
(A) At High Magnetic Field(TROSY line-narrowing
effect)
(B) At Low Magnetic Field(almost no TROSY line-narrowing effect)
•DD relaxation is field-independent. However, CSA relaxation B02,
therefore at high magnetic fields, CSA relaxation can be comparable to
DD relaxation, and the interference effect on relaxation can be observed.
Interference between DD and CSA Relaxation Interference between DD and CSA Relaxation
Linewidth
Magnetic field strength
800 (kDa)
150 kDa
50 kDa
•Optimal field strength: 1 GHz for amide NH; 600 MHz for CH in aromatic moieties (500-800 MHz applicable).
TROSY Effect is Field Dependent and Motion Dependent TROSY Effect is Field Dependent and Motion Dependent
Deuteration- NMR structural study of larger proteinsDeuteration- NMR structural study of larger proteins
Deuteration is also an important techniques for NMR study of larger proteins (> 20kDa). It is achieved by raising the E. coli. in D2O medium (NT$ 10,000 / 1L D2O).
Because of the significantly lower gyromagnetic ratio of 2H compared to 1H ([2H] / [1
H] = 0.15), replacement of protons with deuterons removes contributions to proton linewidths from proton-proton dipolar relaxation and 1H-1H scalar couplings.
The effect of deuteration is similar with that of TROSY and both techniques are frequently used for NMR study of larger proteins.
2H,15N-Gyrase-45 (45 kDa), 750 MHz
Current Opinion in Structural Biology (1999) 9, p,594-601.
The Sensitivity and Resolution Gain by The Sensitivity and Resolution Gain by TROSY and DeuterationTROSY and Deuteration
Paramagnetic spin labeling
MTSL
Paramagnetic Relaxation Enhancement (PRE)Paramagnetic Relaxation Enhancement (PRE)
