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Transcript of DU NMR 4 - University of Denvermysite.du.edu/~balzar/DU_NMR_4.pdf · size limit ~ 30 kDa isotope...
1
Tatiana KutateladzeTatiana Kutateladze
Department of PharmacologyDepartment of PharmacologyUniversity of Colorado Denver University of Colorado Denver
AnschutzAnschutz Medical CampusMedical Campus
Biological applications of NMR Biological applications of NMR spectroscopyspectroscopy
Suggested textsSuggested texts
Protein NMR Spectroscopy: Principle and practice Protein NMR Spectroscopy: Principle and practice –– J. J. CavanaghCavanaghW. W. FairbrotherFairbrother, A. Palmer III and N. Skelton, A. Palmer III and N. Skelton
NMR of Proteins and Nucleic Acids NMR of Proteins and Nucleic Acids –– K. K. WWüüthrichthrich
Biomolecular NMR Spectroscopy Biomolecular NMR Spectroscopy –– J. EvansJ. Evans
NMR of Proteins NMR of Proteins –– edited by G. edited by G. CloreClore and A. and A. GronenbornGronenborn
Introduction to Protein Structure Introduction to Protein Structure –– C. C. BrandenBranden and J. and J. ToozeTooze
Spin Dynamics Spin Dynamics –– M. M. LevittLevitt
Modern NMR Techniques for Chemistry Research Modern NMR Techniques for Chemistry Research –– A. A. DeromeDeromeA complete introduction to modern NMR spectroscopy A complete introduction to modern NMR spectroscopy –– R. R. MacomberMacomber
Protein Data Bank: www.rcsb.org/pdb/index.htmlResearch Collaboration for Structural Bioinformatics -Rutgers U., UC San Diego, NIST
Total 50,830 structures (17 May 2008)41,013 structures (9 January 2007)18,359 structures (1 August 2002)3,298 structures in 2001~90% - crystal; ~9% - solution structures
BioMagResBank: www.bmrb.wisc.edu/Welcome.htmlUniversity of Wisconsin-Madison
3,940 – proteins/peptides chemical shifts123 – DNA coupling constants81 - RNA NMR software library
pulse sequence libraryreference information
StructureStructure--Based Drug Design is the major Based Drug Design is the major objective of structural biologyobjective of structural biology
the use of atomic resolution structural information the use of atomic resolution structural information about a macromolecule typically obtained by Xabout a macromolecule typically obtained by X--ray ray crystallography or NMR spectroscopycrystallography or NMR spectroscopy
the 3D structure of a target protein bound to a lead the 3D structure of a target protein bound to a lead compound allows the atomcompound allows the atom--byby--atom modification and atom modification and improvement of drug leadsimprovement of drug leads
the process is iterative, with successive cycles of the process is iterative, with successive cycles of design, synthesis and testing that refine the properties of design, synthesis and testing that refine the properties of the drug leadthe drug lead
The structureThe structure--based drug design cyclebased drug design cycle
Lead GenerationLead GenerationLead Generation
Chemical SynthesisChemical SynthesisChemical Synthesis
Biological TestingBiological TestingBiological Testing
Pre-clinical StudiesPrePre--clinical Studiesclinical Studies
if promising
Molecular BiologyMolecular BiologyMolecular Biology
Analog DesignAnalog DesignAnalog Design
3D Structure:Crystallography
NMRHomology modeling
3D Structure:3D Structure:CrystallographyCrystallography
NMRNMRHomology modelingHomology modeling
Protein ChemistryProtein ChemistryProtein Chemistry
The structural biology methodsThe structural biology methods
NMR spectroscopyNMR spectroscopyXX--ray crystallographyray crystallographyElectron microscopyElectron microscopyMass spectrometryMass spectrometryCD spectroscopyCD spectroscopyIR IR UV UV EPREPRMolecular modeling and structure predictionMolecular modeling and structure prediction
2
NMR applications and limitationsNMR applications and limitations
time consumingcomplexlow sensitivity (requires 10 mg of a protein)solubility (mM concentrations)size limit ~ 30 kDaisotope labeling (13C, 15N, 2H) is required for large proteins but can be difficult and expensive
in solution - native statedynamicskineticsfoldingintermolecular interactionschemical characterization and analytical validation of compoundsdetection of impurities
What do we What do we start with?start with?
1. Instrument1. Instrument
900 MHz900 MHzNMRNMRspectrometerspectrometer
4.54.5--5.0 cm5.0 cm
NMR sampleNMR sample
550 550 µµll of 0.1of 0.1--2 2 mMmM unlabeled, unlabeled, 1515NN--oror 1515N/N/1313CC-- isotope labeled proteinisotope labeled proteindeuterateddeuterated or phosphate bufferor phosphate bufferpH 2pH 2--77at least 95% purityat least 95% puritynot aggregatednot aggregatedstable for several days/weeksstable for several days/weeksdd1010--DTT (DTT (CysCys))NaNNaN33
Strategy to determine the protein structure Strategy to determine the protein structure by NMRby NMR
1
2
3
4
5
67
Functional assays
2 mo
Functional assays
2 mo
Amino acid analysis, secondary structure by CDand 1H NMR, integrity and
purity by mass spectrometry2 weeks
Amino acid analysis, secondary structure by CDand 1H NMR, integrity and
purity by mass spectrometry2 weeks
Structure calculation andrefinement
2-3 mo
Structure calculation andrefinement
2-3 mo
Oligomeric state by NMR and sedimentation ultracentrifugation
1 week
Oligomeric state by NMR and sedimentation ultracentrifugation
1 week
Analysis of the NMR data:• Sequence specific resonance assignment• NOEs (distance restraints)• Coupling constants (torsion angle restraints)• Exchange rate• Secondary structure
6 mo
Analysis of the NMR data:• Sequence specific resonance assignment• NOEs (distance restraints)• Coupling constants (torsion angle restraints)• Exchange rate• Secondary structure
6 mo
Protein purification and Optimization of the conditions
2 mo
Protein purification and Optimization of the conditions
2 mo
Collection of NMR spectra1-2 mo
Collection of NMR spectra1-2 mo
Dynamicsstudy
2 mo
Dynamicsstudy
2 mo
8
NMR experimentsNMR experimentsNMR experiments
Sequential assignment of amino acidsSequential assignment of amino acidsintraresidueintraresidue=3D HNCACB=3D HNCACB=3D HNHA=3D HNHAinterresidueinterresidue=3D CBCA(CO)NH=3D CBCA(CO)NH=3D HNCO=3D HNCO=3D H(CCO)NH TOCSY=3D H(CCO)NH TOCSY=3D C(CO)NH TOCSY=3D C(CO)NH TOCSY
Identification of amino acid spin system and chemical shift assiIdentification of amino acid spin system and chemical shift assignmentgnment= 2D homonuclear TOCSY= 2D homonuclear TOCSY= 2D homonuclear COSY = 2D homonuclear COSY
backbonebackbone sidechainsidechain=2D =2D 1515NN--edited HSQCedited HSQC =3D =3D 1515NN--edited DIPSIedited DIPSI--HSQC HSQC =2D =2D 1313CC--edited HSQCedited HSQC =3D =3D 1313CC--edited HCCH TOCSYedited HCCH TOCSY
=2D HBCBCGCDHD TOCSY=2D HBCBCGCDHD TOCSY=2D HBCBCGCDCEHE TOCSY=2D HBCBCGCDCEHE TOCSY
NOENOE’’ss assignmentassignment=2D homonuclear NOESY=2D homonuclear NOESY=3D =3D 1515NN--edited NOESYedited NOESY--HSQCHSQC=3D =3D 1313CC--edited NOESYedited NOESY--HSQCHSQC=4D =4D 1313CC--edited NOESYedited NOESY--HSQCHSQC=3D HSQC=3D HSQC--NOESYNOESY--HSQCHSQC=3D =3D 1515N,N,1313CC--edited NOESYedited NOESY
33JJHNHHNHαα coupling constantscoupling constants=3D HNHA=3D HNHA =2D HMQC=2D HMQC--JJ
Total experiment time 103 hrsTotal experiment time 10Total experiment time 1033 hrshrs
NMR experimentsNMR experiments
COSYCOSY:: correlation spectroscopy, provides through bond correlation spectroscopy, provides through bond connections for protons separated by 2 or 3 bondsconnections for protons separated by 2 or 3 bonds
TOCSY:TOCSY: total correlation spectroscopy, provides through bond total correlation spectroscopy, provides through bond connections for all protons in an amino acid side chainconnections for all protons in an amino acid side chain
NOESY:NOESY: nuclear Overhauser effect spectroscopy, provides nuclear Overhauser effect spectroscopy, provides through space connections between protons that are close in the through space connections between protons that are close in the spacespace
HSQC:HSQC: heteronuclear single quantum coherence, provides heteronuclear single quantum coherence, provides connections between a proton and a directly attached connections between a proton and a directly attached 1515N or N or 1313C nucleiC nuclei
3
NMR parametersNMR parameters
Chemical shift: Chemical shift: δ δ (corresponds to the resonance frequency of a nucleus; (corresponds to the resonance frequency of a nucleus; related to the chemical environment of the nucleus; affected by related to the chemical environment of the nucleus; affected by aromatic ring currents, aromatic ring currents, solvent exchange and solvent exchange and electronegativityelectronegativity of nearby nuclei; units: (of nearby nuclei; units: (ppmppm) parts per million)) parts per million)
SpinSpin--spin coupling constant: J spin coupling constant: J (characterizes through(characterizes through--bond bond (scalar) interactions; related to the bond angles; causes NMR li(scalar) interactions; related to the bond angles; causes NMR lines of coupled nuclei to nes of coupled nuclei to split; units: Hz)split; units: Hz)
Nuclear Overhauser effect: NOE Nuclear Overhauser effect: NOE (caused by through(caused by through--space space (dipolar) interactions between nuclei separated in space by less(dipolar) interactions between nuclei separated in space by less than 5 A; detected by than 5 A; detected by changes in NMR line intensity of one nucleus when another is irrchanges in NMR line intensity of one nucleus when another is irradiated; correlated by adiated; correlated by the inverse sixth power of the distance between the nuclei)the inverse sixth power of the distance between the nuclei)
Relaxation parameters: Relaxation parameters: linewidthlinewidth, T, T11 and Tand T2 2 (longitudinal and transverse relaxation times)(longitudinal and transverse relaxation times)
foldedprotein
unfoldedprotein
backbone NH
aromatic
side-chain NH
αH
aliphatic
methyl
10 9 8 7
H1 ppm
130
129
128
127
126
125
124
123
122
121
120
119
118
117
116
115
114
113
112
111
110
109
N15 ppm
10 9 8 7
H1 ppm
130
129
128
127
126
125
124
123
122
121
120
119
118
117
116
115
114
113
112
111
110
109
108
N15 ppm
1D 1H
90 residuesprotein:H : ~650C : ~420N : ~140
1H-15N HSQC
Scalar spin-spin coupling connectivities in TOCSY (total correlation spectroscopy)
a. all nonlabile, nonaromatic side chain 1Hb. αH-βCH3 of Ala, βH-γCH3 of Thrc. αH-βH of Val, Ile, Leu, Gln, Glu, Met,
Pro, Arg and Lysd. αH-βH of Cys, Asp, Asn,
Phe, Tyr, His and Trpe. αH-αH of Gly, αH-βH of Thr,
δH-δH of Pro, αH-βH andβH-βH of Ser
f. aromatic 1H
a
bcde
f
8.0 7.5 7.0 6.5
8.0
7.5
7.0
6.5
Z
Z2
D1D
E3
E1
E
E
D2
D
D2
E1
E1
Z
Z
Z3
H2
D2
D
E
ee42t1 (DIPSI, D2O)Phe42Phe58Phe83
His18His49His50
Trp26
ZED
D1Z2
H2Z3
E3
N
E1
D2N
N
H chemical shift (ppm)1
H chem
ical shift (ppm)
1
Identification of 1H spin systems in aromatic residues
2D NOESY (H2O) shows through space connectivities
βF58
βF58
αF58
NHF58NHA60
αC59
NHC59
αA60
NHE61
dαN, dNN, dβN
AMXAMX
A3X
Identification of spin systems and complete chemical shift assignment
intraresidue: 3D 15N- and 13C-edited TOCSY,Interresidue: 3D HCC and CCC TOCSY, HNCO
Identification of spin systems and complete chemical shift assignment
intraresidue: 3D 15N- and 13C-edited TOCSY,Interresidue: 3D HCC and CCC TOCSY, HNCO
Sequential assignment of amino acids using triple-resonance HNCACB,CBCA(CO)NH + 1H-15N HSQC
Sequential assignment of amino acids using triple-resonance HNCACB,CBCA(CO)NH + 1H-15N HSQC
NOEs: 3D 15N- and 13C-edited NOESY NOEs: 3D 15N- and 13C-edited NOESY
Protein structure determination using 3D and 4Dspectra of 15N/13C labeled proteins
3JHNHα coupling constants: 3D HNHA, HMQC-J3JHNHα coupling constants: 3D HNHA, HMQC-J
10 9 8 7
130
125
120
115
110
A3
N34
G39G55
T69
G41
G8S71
T45
R9
R24
D29
C35
S43
L7
A27
E28
Q33
F42
K17
W26
V32
K40 V46
H49
K25
E31C38 R48
C51
N30
A37
V44
R47
H50
R52M36
Q53
N56
F58A60
C62
C54
I57
C59
E61
S63
A64
K65
N66
A67 L68
S72
K74
V76
R77
V78
C79D80
A81
C82
F83
N84
D85
L86
Q87
G88
Q5
E6
E10
N11
Q12
S13
H18
T19
Q20
A21
L14
Q15
I16
L22
N23
V4
N56
N34
N84N11
Q53
N66Q12
Q20
Q87
Q15
N30
Q5
Q33
N23
W26
N-HSQC, ee44ns15
N ch
em
ical sh
ift (ppm
)15
H chemical shift (ppm)1Structure calculation Structure calculation
4
Sequential resonance assignmentSequential resonance assignment
130
125
120
115
110
N-HMQC-NOESY-HMQC, ee22nnn115
A37 C38 G39 K40 G41 F42 G55C51 R52 Q53 C54R48 H49 H50 N56
-HN-CH-CO-HN-CH-CO-HN-CH-CO- R R R
N ch
em
ical sh
ift (ppm
)1
5
70
60
50
40
30
20
T45 V46 R47R48H49H50 C51R52 Q53C54 G55 N56 I57 F58 C59 A60 E61
ee44nab1 (HNCACB) O C O -C-C-N-C-C- C H
α
β
β
α
C ch
em
ical sh
ift (ppm
)13
Val78Val78
γ1γ1
ββ
γ2γ2ββ
δ1δ1
δ2δ2
γγ
Leu86Leu86ββAla67Ala67
Phe83Phe83δδ
εε
R<5Å
3D 13C-edited NOESY
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
A67 S71 S72T69L68 V78V76K74 R77N66 C82A81D80 L86F83C79 N84 D85
α helix
β4 strandβ3 strand
α
β
γδδ
δδ
β
β
α
α
α
αα
α
γ
γβ
ββ
β
β
β
NH
NH
NHNH
NH
NH
NH
NH
α
γγγ
β
βγ
β
ee22nn2 (NOESY, mix=150 ms)
H ch
em
ical sh
ift (ppm
)1
The secondary The secondary structure structure determination and determination and collection of the collection of the ddNXNX restraints in 3D restraints in 3D 1515NN--edited NOESYedited NOESY
Computational approach to determine the Computational approach to determine the structure of a proteinstructure of a protein
1. known sequence of the amino acid residues1. known sequence of the amino acid residues2. known molecular structure of the residues, including bond dis2. known molecular structure of the residues, including bond distancestances
and anglesand angles3. known planar structure of the peptide group3. known planar structure of the peptide group4. NMR4. NMR--derived restraints:derived restraints:•• Interproton distances (NOEs)Interproton distances (NOEs)
weak, medium and strong intensity weak, medium and strong intensity •• IntraIntra-- and interresidue bond anglesand interresidue bond angles
ΦΦ, , ΨΨ, , nn
•• Hydrogen bondsHydrogen bondsNN--O and HO and H--OO distancesdistances
•• Heteroatom distance and angle restraintsHeteroatom distance and angle restraintsCaCa2+2+, Mg, Mg2+2+, Zn, Zn2+2+, etc., etc.
Input:Input:
Calculation and refinement of the 3D Calculation and refinement of the 3D structurestructure
InputInput
•• Distance geometry Distance geometry •• Restrained molecular dynamics Restrained molecular dynamics
simulated annealingsimulated annealing
Minimization of system energy:Minimization of system energy:bond length and angles,bond length and angles,van der van der WaalsWaalsMinimization of violations:Minimization of violations:NOEs, dihedral anglesNOEs, dihedral anglesSeveral cycles Several cycles -- calculationcalculation--analysis of violated restraintsanalysis of violated restraints
A set of 20 lowest A set of 20 lowest energy structuresenergy structures
AverageAveragestructurestructure
Input restraint summary
Output: Structural statistics
5
Root Mean Square Deviation (RMSD) –a measure of the quality of the NMR structure
The number of restraints effects both precision and accuracy
Precise Accurate Precise andaccurate8 – 15 restraints/res
RMSD = √ Σ (ri - r’i)2N
i=1
1N
Beyond the structureBeyond the structure
rapid screening of lead compounds (SHAPES and other libraries)
dynamic regions: motions of the protein backbone
conformational changes
pre-screening of structural targets for X-ray and NMR
structures of proteins that do not crystallize
structures of multi-domain proteins in solution
determination of small protein structures (cryogenic probes, high field magnets, residual dipolar couplings, TROSY, micelles, automatic resonance assignment and NOE peaks picking)
rapid screening of lead compounds (SHAPES and other libraries)rapid screening of lead compounds (SHAPES and other libraries)
dynamic regions: motions of the protein backbonedynamic regions: motions of the protein backbone
conformational changesconformational changes
prepre--screening of structural targets for Xscreening of structural targets for X--ray and NMRray and NMR
structures of proteins that do not crystallizestructures of proteins that do not crystallize
structures of multistructures of multi--domain proteins in solutiondomain proteins in solution
determination of small protein structures (cryogenic probes, higdetermination of small protein structures (cryogenic probes, high field h field magnets, residual dipolar couplings, TROSY, micelles, automatic magnets, residual dipolar couplings, TROSY, micelles, automatic resonance resonance assignment and NOE peaks picking)assignment and NOE peaks picking)
qualitative and quantitative binding assay: changes in NMR paramqualitative and quantitative binding assay: changes in NMR parameters allow eters allow us to determine a us to determine a ligandligand’’ss binding site and to estimate binding affinitiesbinding site and to estimate binding affinities
NMRNMR--based methods to monitor based methods to monitor proteinprotein--ligand interactionsligand interactions
chemical shift perturbationschemical shift perturbationsintermolecular NOEsintermolecular NOEsTT11/T/T22 relaxationrelaxation
9 8 7130
125
120
115
110
N c
hem
ical
shi
ft (p
pm)
15
H chemical shift (ppm)1
S1365
R1369
R1374
A1382
Q1375
A1389
F1364
I1379R1370E1353
C1373
A1386R1399
K1347
N1378
H1372
N1352
0.3 0.3 0.3 0.3
[PtdIns(3)P]0.0 mM0.1 0.2 0.3 0.4 0.5
F1380
M1358
Q1355
HO O
O
O
ORCOOP
OHOH
OHO PO3
=
-RCOO
A,V,L,I,P,M,F,W
K,R,HD,E
T,S
Mapping the binding site residuesbased on the chemical shift perturbationsbased on the chemical shift perturbations
Mapping the binding site of a ligand
advantages: fast, low protein advantages: fast, low protein concentration, even weak binding, no concentration, even weak binding, no false positivefalse positive
The HSQC titration experiments provide quick The HSQC titration experiments provide quick information on the affinityinformation on the affinity
6
NMRNMR--based methods to monitor based methods to monitor proteinprotein--ligand interactionsligand interactions
chemical shift perturbationschemical shift perturbations
TT11/T/T22 relaxation changesrelaxation changesintermolecular NOEsintermolecular NOEs
Labeling with stable Labeling with stable isotopes, such as isotopes, such as 1515N, N, 1313C and/or C and/or 22H H together with together with ‘‘isotope editingisotope editing’’experiments allows experiments allows us to select signalsus to select signals
Intermolecular NOEs – interproton distances between two molecules