DU NMR 4 - University of Denvermysite.du.edu/~balzar/DU_NMR_4.pdf · size limit ~ 30 kDa isotope...

6
1 Tatiana Kutateladze Tatiana Kutateladze Department of Pharmacology Department of Pharmacology University of Colorado Denver University of Colorado Denver Anschutz Anschutz Medical Campus Medical Campus Biological applications of NMR Biological applications of NMR spectroscopy spectroscopy Suggested texts Suggested texts Protein NMR Spectroscopy: Principle and practice Protein NMR Spectroscopy: Principle and practice – J. J. Cavanagh Cavanagh W. W. Fairbrother Fairbrother, 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. Wüthrich thrich Biomolecular NMR Spectroscopy Biomolecular NMR Spectroscopy – J. Evans J. Evans NMR of Proteins NMR of Proteins – edited by G. edited by G. Clore Clore and A. and A. Gronenborn Gronenborn Introduction to Protein Structure Introduction to Protein Structure – C. C. Branden Branden and J. and J. Tooze Tooze Spin Dynamics Spin Dynamics – M. M. Levitt Levitt Modern NMR Techniques for Chemistry Research Modern NMR Techniques for Chemistry Research – A. A. Derome Derome A complete introduction to modern NMR spectroscopy A complete introduction to modern NMR spectroscopy – R. R. Macomber Macomber Protein Data Bank: www.rcsb.org/pdb/index.html Research 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.html University of Wisconsin-Madison 3,940 – proteins/peptides chemical shifts 123 – DNA coupling constants 81 - RNA NMR software library pulse sequence library reference information Structure Structure-Based Drug Design is the major Based Drug Design is the major objective of structural biology objective of structural biology the use of atomic resolution structural information the use of atomic resolution structural information about a macromolecule typically obtained by X about a macromolecule typically obtained by X-ray ray crystallography or NMR spectroscopy crystallography 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 atom compound allows the atom-by by-atom modification and atom modification and improvement of drug leads improvement 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 lead the drug lead The structure The structure-based drug design cycle based drug design cycle Lead Generation Lead Generation Lead Generation Chemical Synthesis Chemical Synthesis Chemical Synthesis Biological Testing Biological Testing Biological Testing Pre-clinical Studies Pre Pre-clinical Studies clinical Studies if promising Molecular Biology Molecular Biology Molecular Biology Analog Design Analog Design Analog Design 3D Structure: Crystallography NMR Homology modeling 3D Structure: 3D Structure: Crystallography Crystallography NMR NMR Homology modeling Homology modeling Protein Chemistry Protein Chemistry Protein Chemistry The structural biology methods The structural biology methods NMR spectroscopy NMR spectroscopy X-ray crystallography ray crystallography Electron microscopy Electron microscopy Mass spectrometry Mass spectrometry CD spectroscopy CD spectroscopy IR IR UV UV EPR EPR Molecular modeling and structure prediction Molecular modeling and structure prediction

Transcript of DU NMR 4 - University of Denvermysite.du.edu/~balzar/DU_NMR_4.pdf · size limit ~ 30 kDa isotope...

Page 1: DU NMR 4 - University of Denvermysite.du.edu/~balzar/DU_NMR_4.pdf · size limit ~ 30 kDa isotope labeling (13C, 15N, 2H) ... or15N/N/13CC-- isotope labeled protein ... the inverse

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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

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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

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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

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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

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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

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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