PDB Composition (2003)

Transcription

1 Biomolecular NMR Dr. Kiattawee hoowongkomon Dept. of Biochemistry Faculty of Science Kasetsart University Phone: ext. 121

2 PDB omposition (2003) Proteins Protein/DNA complexes DNA/RNA X-ray (84%) 636 (86%) 602 (57%) NMR 2174 (14%) 82 (11%) 424 (40%) Models 321 (2%) 24 (3%) 8 (3%)

3 Principles of NMR Measures nuclear magnetism or changes in nuclear magnetism in a molecule NMR spectroscopy measures the absorption of radio waves due to changes in nuclear spin orientation NMR only occurs when a sample is in a strong magnetic field Different nuclei absorb at different energies (frequencies)

4 Principles of NMR

5 Principles of NMR N N hν S Low Energy S igh Energy

6 The Bell Analogy 3 N

7 Intrinsic Sensitivity of Nuclei Nucleus γ % Natural Relative Abundance Sensitivity x x N -2.7 x P 1.1 x Prepare samples enriched in these nuclei

8 The spin-lattice relaxation time T1 The return to equilibrium along the z-axis is the T1

9 The spin-spin relaxation time T2 Following a 90º pulse The de-phasing of this precession is mediated by the spin-spin relaxation T2

10 FT NMR Free Induction Decay FT NMR spectrum

11 Information from NMR hemical Shifts A variation in the resonance frequency of a nuclear spin due to the chemical environment around the nucleus (in ppm) 1, 15 N, 13 can be observed in proteins Nuclear Overhauser Effects (NOEs) A result of cross-relaxation relaxation between dipolar coupled spins interaction through space. Distance information through space 5 Å NOE 1/r 6 J coupling constants J coupling is mediated through chemical bonds connecting two spins orrelated to backbone dihedral angle 3 J αn

12 Regions of the 1 NMR Spectrum are Further Dispersed by the 3D Fold

13 The Pulse FT NMR Experiment Experiment 90º pulse (t) equilibration detection of signals Data Analysis Fourier Transform Time domain (t)

14 One-dimension 1 NMR spectra EGFR EGFR Micelle 25 Micelle 25 Micelle 35 Micelle 35 RRRIV RKRTLRRLLQ ERELVEPLTP SG-N2 RLLQ ERELVEPLTP SGEA

15 2D NMR: oupling is the Key 90º pulse 2D detect signals twice (before/after coupling) Same as 1D experiment Transfers between coupled spins

16 The 2D NMR Spectrum Pulse Sequence Spectrum t1 t2 Before mixing oupled spins After mixing

17 The Power of 2D NMR: Resolving Overlapping Signals 1D 2 signals overlapped 2D 2 cross peaks resolved

18 igher Dimensional NMR: Built on the 2D Principle 90º pulse 3D- detect signals 3 times (t3) Same as 1D experiment

19 2-D D NMR: TOSY TOtal orrelation SpectroscopY TOSY is an relayed extension of OSY uses scalar coupling ross-peaks appear between all spins which can be connected by relaying Magnetisation still can t t be transferred across peptide bond (3-bond limit still applies) amino acids still form isolated spin systems Useful for recognising particular amino acids

20 One-dimension 1 NMR spectra EGFR EGFR Micelle 25 Micelle 25 Micelle 35 Micelle 35 RRRIV RKRTLRRLLQ ERELVEPLTP SG-N2 RLLQ ERELVEPLTP SGEA

21 TOSY EGFR EGFR

22 2-D D NMR: OSY OSY(correlation spectroscopy)/jcoupling Jcoupling: protons that are bonded to each other can be directly spin-coupled; can track one atom to the next

23 0 OSY hemical Shift ppm 1 hemical Shift

24 0 OSY hemical Shift ppm 1 hemical Shift

25 0 OSY hemical Shift ppm 1 hemical Shift

26 2-D D NMR: NOESY NOESY(nuclear Overhauser effect spectroscopy)/noe coupling : protons closer than 0.5 nm will perturb each others spins even if they are not closely coupled in the primary structure; spatial determination

27 TOSY NOESY Leu Ala Asn Gly N Leu Ala Asn Gly N In the TOSY we see all the spins. The NOESY will have both intraresidue correlations ( ), as well as interesidue correlations ( ), which allows to find which residue is next to which.

28 Tocsy and Noesy overlay

29 Tocsy and Noesy only Amine Region

30 eteronuclear NMR Peptide bond 3-bond limit means that cross- peaks are never observed between protons in different amino acids; i.e. there is no magnetization transfer across the peptide bond Magnetization can be transferred if the intervening nuclei are magnetic; i.e. 13 and 15 N. This is achieved by producing the protein recombinantly in bacteria grown with 15 N-ammonium chloride and 13 -glucose as the sole nitrogen and carbon sources respectively

31 Double-Resonance Experiments Increases Resolution/Information ontent 15 N- 1 SQ R R - 15 N - α -O - 15 N - α

32 Resolve Peaks By Multi-D D NMR If 2D cross peaks overlap go to 3D or 4D..

33 3D experiments 90º pulse 3D- detect signals 3 times (t3) Same as 1D experiment ω N ω α The previous experiments can be extended to two indirect dimensions, t 1 and t 2 The real time interval during which all the FID s are recorded is called t 3, or the direct dimension. S is a function of t 1, t 2, and t 3 ; to get the spectrum it must be Fourier transformed inall three time dimensions. If the magnetization is on a nucleus with frequency ω 1 in t 1, ω 2 in t 2 and ω 3 in t 3, the spectrum will have a peak centred at coordinates (ω( 1, ω 2, ω 3 ) In 3D a peak is more like a ball ω N

34 Pulse sequence for measuring Γ αα, 1 Δ 2 Δ 2 y x y Δ DIPSI-2(x) -y y DIPSI-2(x) -y Δ Δ 2 Δ 2 y Δ 2 Δ 2 φ rec t3 15 N τ τ τ _ t 2 2 τ t 2 2 Ψ GARP 13' δ Δ' 2 2 δ_ 2 Δ' 2 Τ_ 4 _ Δ' 4 Τ_ + 4 Δ' 4 Τ_ + 4 Δ' 4 Τ 4 φ6 -y δ t _ 1 δ_ Δ' 4 t 1 _ 2 13 α α BSP PFG G1 G2 G3 G4 G3 G4 G5 G6 κg7 G7 Reference experiment: φ6 = y, Δ' = 0 "ross" experiment: φ6 = x, Δ' = 1/[2J( α, α)]

35 eteronuclear assignment experiments residue i-1 residue i 3D NA experiment protein must be isotopically enriched with 1, 13 and 15 N Peaks represented as balls in 3D space at coordinates corresponding to: 1 shift of an amide proton ( N ) 15 N shift of attached N 13 shift of attached α At same 1 and 15 N values, another peak corresponding to 13 shift of α of preceding residue makes it possible to walk along sequence to assign entire backbone

36 Assignment based on J-correlationsJ

37 Relative sensitivity of triple resonance experiments Experiment Assignment omment Relative S/N [%] NO (i), N(i), (i-1) <20 kd, above use 2 labeling 100 NA (i), N(i), α (i), α (i-1) <20 kd, above use 2 labeling 50/15 N(O)A (i), N(i), α (i-1) <20 kd, above use 2 labeling 71 N(A)O (i), N(i), (i) <20 kd, above use 2 labeling 13/4 BA(O)N (i), N(i), α (i-1), β (i-1) <20 kd, above use 2 labeling 13/9 α/β BA(O)N (i), N(i), α (i-1), β (i-1) <20 kd, above use 2 labeling 13/9 α/β BAN, NAB ()(O)N- TOSY (i), N(i), α (i), β (i), α (i-1), β (i-1) (i), N(i), aliph. (i-1) <15 kd, above use 2 labeling 4/1.7 α/β(i) 1.3/0.5 α/β(i-1) <15-20 kd, above use 2 labeling ()(O)N- (i), N(i), aliph. (i-1) <15-20 kd, above use 2 labeling TOSY -TOSY aliph., aliph. <25 kd, - sensitive, but tedious to analyze, combine with ON type experiments

38 Typical NMR structure determination of a small protein domain Experiment Time Software Information 3D NA 2.5d NMRPIPE backbone 3D BAON 2d - - chemical shift 3D NAB 2.5d - - assignments Backbone assignment 2weeks XEASY 3D ()ON-TOSY 2.5d NMRPIPE side chain 3D ()ON-TOSY 2.5d - - chemical shift 3D -TOSY 3d - - assignments Side chain assignment 2weeks XEASY 2D NOESY 1d NMRPIPE assignments + 3D 15 N-edited NOESY 3-4d - - NOE derived 3D 13 -edited NOESY 3-4d - - distance restraints Distance restraints/ 4-8weeks XEASY Structure calculation 4-8weeks ARIA/NS 3D NA-J 3d scripts φ angles 2D NG (aliph./arom.) 1d scripts χ 1 angles Dihedral angles 2d 2D α/β correlation ( N -N) 0.5d XEASY projection angles Residual dipolar couplings 1d scripts /D exchange 1d scripts hydrogen bonds TOTAL TIME 2-5 months

39 Assignment Strategy Interactions through bond Backbone NO, N(A)O NA, NAB BA(O)N Interactions through space 15 N/ 13 -SQ-NOESY Sidechain (O)N (O)N ()-TOSY onnection through bond = shapes of jigsaw puzzles onnection through space = pictures on jigsaw puzzles

40 Sequence Assignments via hetero nuclear Experiment NA NOA NOAB SQ-TOSY N N N N

41 N N N N 1-15 N SQ 15 N 2D SQ yields one resonance for each amide N

42 N N N 1-15 N SQ NA N N

43 N N NA 1 13 NA experiment yields a cross peak between the N proton and the α in the same amino acid and from the previous amino acid N SQ 15 N

44 NOA N N 13 1 NOA experiment yields a cross peak between the N proton and only the α from the previous amino acid. 15 N

45 NA N N 13 1 ombining the results from the NA and NOA experiments allows one to assign the N..α cross peak for each amino acid. 15 N

46 4 < < < < 3 < < 2 < 5 ω 1 < ω N ω N Assignment of all N, N and α resonances of a pentapeptide in a NA spectrum by walking along the backbone. In each case the black sphere represents the in-residue α, the grey sphere the α of the preceding residue

47 eteronuclear Assignments. Intra-Residue vs. Sequential: NA and N(O)A Experiments β β β -N- α - ο -N- α - ο -N- α - ο - NA N(O)A

48 eteronuclear Resonance Assignments: Sequential Assignment Walk 15 N 1 13 Figure source: J. Markley (University of Madison-Wisconsin). NMR notes: used without permission

49 eteronuclear Assignments. Intra-Residue vs. Sequential: NAB and BA(O)N Experiments β β β -N- α - ο -N- α - ο -N- α - ο - NAB BA(O)N

50 NOAB N N 13 1 NOAB experiments allows one to assign the N..α and β cross peak for the previous amino acid. In this case, four alanines are resolved. 15 N

51 NAB N N 13 NAB experiments yields cross peaks between the N proton and the α and β in the same amino acid and from the previous amino acid N 1

52 NAB N N 13 ompare NOAB and NAB experiments for assignments N

53 NAB β α

54 The NMR Process Obtain protein sequence ollect TOSY & NOESY data Use chemical shift tables and known sequence to assign TOSY spectrum Use TOSY to assign NOESY spectrum Obtain inter and intra-residue residue distance information from NOESY data Feed data to computer to solve structure

55 Information from NMR hemical Shifts A variation in the resonance frequency of a nuclear spin due to the chemical environment around the nucleus (in ppm) 1, 15 N, 13 can be observed in proteins Nuclear Overhauser Effects (NOEs) A result of cross-relaxation relaxation between dipolar coupled spins interaction through space. Distance information through space 5 Å NOE 1/r 6 J coupling constants J coupling is mediated through chemical bonds connecting two spins orrelated to backbone dihedral angle 3 J αn

56 Secondary Structure Indicators Secondary chemical shifts (Δδ( Δδ) alculate from different between the observed and random coil database chemical shifts of each amino acid Statistic distribution can use to identify the secondary structures α β O α α-helix positive negative positive negative β-sheet negative positive negative positive Wishart, et al., Biochemistry, 31, 1647 (1992) Wishart, et al., J. Biomol. NMR, 4, 171 (1994)

57 Secondary chemical shifts elix Beta-sheet

58 NOE effect provides structural information Nuclear Overhauser Effect produces coupling between protons which are close in space (though not necessarily covalently bonded) NOE cross-peaks R -6 only observed for R < 5 Å NOESY is 2D experiment in which cross peak intensities are proportional to NOE between corresponding protons NOESY spectrum of lysozyme

59 Secondary Structure Indicators Medium-range NOEs α-helix Å β-sheet (i) (i+3) (i+6) 4.2 Å (i+4) 2.2 Å

60 Tertiary Structure NOE Long-range NOE pattern NOE NOE NOE NOE Intensity Weak Medium Strong Distance Å Å Å

61 haracteristic NOE patterns. The easiest to identify are interesidue and sequential NOE, cross-peaks, which are NOEs among protons of the same residue and from a residue to protons of the (i + 1) and (i - 1) residues: d αn d αα d NN O AA 2 α O N N N N d Nβ, d Nγ, AA 1 α O AA 3 α dαβ, d αγ, d αα

62 Apart from those, regular secondary structure will have regular NOE patterns. For a-helices and b-sheets we have: i+4 d αβ(i, i+3) d αn(i, i+3) d NN(i, i+3) d αn (i, i+4) i-1 i i+3 i+2 i+1 N N d α(i)n(j) d α(i)α(j) d N(i)N(j) N N

63 Strategies for Sequential Assignment Problem: there are a few proline residues in most proteins. Problem: there are a number of additional short proton proton distances which can occur as a result of certain elements of secondary structure. The general work of Wuthrich and co-workers identified a whole range of secondary specific short proton proton distances that are summarized here:

64 Must accommodate multiple solutions multiple J values But database shows few occupy higher energy conformations Dihedral Angles From Scalar ouplings 6 z

65 NMR Spectroscopy hemical Shift Assignments NOE Intensities J-ouplings Distance Geometry Simulated Annealing

66 NMR Experimental Observables Providing Structural Information Backbone conformation from chemical shifts (hemical Shift Index- SI): ψ,φ Distance restraints from NOEs ydrogen bond restraints Backbone and side chain dihedral angle restraints from scalar couplings Orientation restraints from residual dipolar couplings

67 1 15 N 13 chemical shift assignments Acquisition of 3D- 13 / 15 N-NOESY- SQ experiments Find NOE assignments Evaluate NOE assignments Structure alculation (NS) J NA -coupling restraints Talos Dihedral restraints RD restraints ompleteness NOE assignment 3D structure

68 Long Range NOE Evaluate NOE assignments Find NOE assignments Structure alculation Long Range NOE + ompleteness Refinement NOE assignment For helical 3D structure domains + Find NOE assignments Evaluate NOE assignments Structure alculation

69 Evaluation riteria Low total energy E total = E bond + E angle + E dihedr + E vdw + E coulomb + E NMR igh E total are came from E NMR (mostly from NOE) that cannot be fulfilled the calculated structures Goal is to get less energy violation from input restraints Recheck the violated NOE Wrong assignment? Ambiguous? Wrong calibration? Evaluate NOE assignments Find NOE assignments Structure alculation

70 Some Real World Examples The hromodomain Assignment of secondary structure of the chromodomain Tertiary structure determined by adding long range NOEs. Ball et al., EMBO J. 16, 2473 (1998)

71 Design of JX-EGFR Peptide EX TM JX TK -terminal 645-RRRIVRKRTLRRLLQERELVEPLTP SGEAPNQALLRILKETE FKKIKVLGSG-697 E. oli (BL21) GST-Met- -is-tag 15 N and 13 sources ( 15 N-NN 4 l 13 -D-Glucose) 15 N/ is-tag (5 mg/ liter)

72 Methods Uniformly 15 N and/or 13 - labeled peptides Pulse sequences Magnet Structure Analysis, Assignment, and Structure alculation NMR Spectra

73 1-15 N-correlation spectra of 15 N-JX Peptide in Water 15 N 645-RRRIVRKRT LRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSG-697 1

74 Secondary Structure Indicators Secondary Structure Indicators elical? Δδ(α) (ppm)

75 Micelles Dodecylphosphocholine (DP) - Membrane mimicking environment -Forming micelles (60 DPs per micelle) -Size of micelle is 40 Å -Fast tumbling

76 1-15 N-correlation Spectra of 15 N-JX in DP Micelles 15 N Water DP RRRIVRKRT LRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSG-697

77 Secondary Structure Indicators Secondary Structure Indicators DP Micelles Δδ(α) (ppm) helical helical helical Water Δδ(α) (ppm)

78

79

80 Refinement Paramagnetic broadening Identification of helixes/ micelle interface Residual dipolar couplings (RD) Relative orientation between helical axes

81 Orientation of elices on Micelle Paramagnetic probes Elements of compound that has a unpaired electron which produces local fluctuating magnetic fields Mnl 2 : water soluble molecule Deoxyl-stearic acid (DSA) : hydrophobic molecule 5-DSA Mn 2+ 5-DSA Mn 2+ N N Mn 2+ 5-DSA 5-DSA

82 Paramagnetic Broadening Effects N676 T N correlation spectra of 15 N-JX in DP micelles mm Mnl mm 5-DSA N- N676 N- T654

83 Paramagnetic Broadening Effects 100 INTENSITY RETENTION (%) A) INTENSITY RETENTION (%) B) Mn DSA RESIDUE

84 Residual Dipolar oupling (RD) Reports angle of inter-nuclear (N-) vector relative to magnetic field B 0 B 0 θ 1 A zz 1 15 N r Each N- vector in the structure much share one common alignment tensor. z A yy 15 N A xx A zz A zz z A yy A zz z A xx yy z A yy A xx A zz z A xx A yy A xx

85 z Residual Dipolar oupling (RD) Residual Dipolar oupling (RD) A zz z Axx A yy A xx A xx z A zz A xx A zz A yy A zz A xx A yy A xx A yy A xx A zz A yy UP DOWN RIGT LEFT Azz z Ayy A zz z A yy

86

87 Residual dipolar coupling (RD) TROSY-SQ in solution TROSY-SQ in compressed gel z z z z

88 Residual Dipolar oupling A) N- RDs (z) RESIDUE

89 Structure Refinement RD elical orientations

90 Location of sorting signals 667-PXXP dominant basolateral signal is in the flexible loop accessible 658-LL recessive basolateral and 679-LL lysosomal signals are in the helical structures not accessible, adsorption on the surface of micelle

91 Physiological Significances Trans Golgi Network Sorting Endosomes Late Endosomes Lysosomes

92 Nuclear Magnetic Resonance in Biology NMR Spectrometer Superconducting magnet: aligning nuclear magnets NMR sample: ontains material for analysis in a special NMR tube Radiofrequency console: exciting and detecting the transitions between different energy levels omputer workstation: data collection and analysis NMR probe: holding sample; irradiating and detecting the radiofrequency signals coming from nuclei

93 Protein NMR Spectroscopy: ritical Features Tertiary structure leads to increased dispersion of resonances Example: 1-15 N SQ spectra of an unfolded vs. folded protein Figure source: J. Markley (University of Madison-Wisconsin). NMR notes: used without permission

94 Amide Exchange /D exchange 15 N- 1 SQ Add D 2 0 and collect time series of spectra

95 mobile, flexible chain is more exposed to solvent and will exchange faster D 2 0 D 2 0 N D N D N D N N D 2 0 D N

96 2D QS as Probes of Protein-Ligand Interactions A key feature of this type of experiment is the natural abundance of NMR relevant isotopes. To perform heteronuclear experiments at natural abundance one needs an extremely high sample concentration. Alternatively, the sample can be enriched with the isotope in question. Isotopic enrichment of proteins with 15 N and 13 by way of bacterial overexpression in minimal media is routine in most NMR laboratories. If a mixture of proteins or protein/peptide or in most general terms protein and ligand are studied then selective isotopic enrichment allows very elegant experiments

97 Isotopic Enrichment for 2D QS Only the protein or the ligand that is enriched in the relevant isotope - here 15 N - is visible in the 15 N- 1 2D SQ experiment. The selective removal of one binding partner will considerably simplify the spectra. The two interaction partners - a big protein and a smallish ligand - are displayed in blue and red, respectively. When a binding partner is enriched in 15 N the color is strong, if not it is pale. In a 15 N experiment, e.g. SQ, only the 15 N component is visible. So if only the protein is labelled with 15 N as indicated in the picture on the right, then it is possible to add arbitrary amounts of ligand without interfering with the quality of the spectrum.

98 3D-15N/13-filtered NOESY experiment Fernandez et. al., PNAS (2002), 99(21),p Vinogradova et. al., PNAS,(2004), 101(12),p. 4094

99 am binding regions Titration of 15 N-JX peptide with calmodulin Broaden peaks Low affinity K D ~ 0.4 μm annot determine the structure 1:0 (peptide :am) 1:3 645-RRRIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSG-697

100 Titration of 15 N-almodulin with EGFR peptide 1:0 (am:peptide) 1:0.5 1:1 1:2 1:3 Precipitation of complex Soluble by DP

101 Interaction of juxtmamenbrane EGFR with S3 domains

102 NKs S3-1 S3-2 S3-3 S2 Rational S3-ligand lass I +xxpxxp lass II PxxPx+ EGFR RELVEPLTPSGE + EGFR peptide coupled to the bead EGFR peptide + Western bolting GST GST Abl rk Fgr Grb2 Nck p85 Spectrin Src - Screening 8 S3 proteins. - ell biology support of Nck - Basolateral localization - Western blot of cell lysate - S3-1 & S3-2? - Nckα and Nckβ? Sample Preparation S3s are easy to unfold (Keith Decker) omputer modeling (Dave) Express GB1 fusion S3 domains of Ncks (Nick)

103 Nckα-1 GB1 fusion proteins Nckα-2 Nckα-3 Nckβ-1 Nckβ-2 Nckβ-3

104 Titration study of Nck N EGFR is-tag (1 mg) + GB1 fusion Nck (1 mg) SQ

105 Alpha-1 Alpha-2 Alpha-3 Beta-1 Beta-2 Beta-3

106 Alpha-1 Alpha-2 Alpha-3 Beta-1 Beta-2 Beta-3

107 Pulsed filed gradient longitudinal eddy-current delay NMR experiment Gounarides et al., 725, 79 (1999). Gao et al., Biophysics,74, 1871 (1998).

108 MDS : hexamethyldisilane Diffusion NMR experiments EGFR EGFR DP SDS DP SDS D free (x 10-7 cm -2 /s) 22.00± 5% 22.00± 5% 16.65± 6% 16.65± 6% D obs (x 10-7 cm -2 /s) 14.12± 4% 5.04± 7% 8.18± 9% 4.93± 5% D micelles (x 10-7 cm -2 /s) 7.46± 2% 5.06± 2% 7.52± 5% 5.20± 8% Bound (%) D obs -D free D micelles -D free MDS + D micelles D free D obs

109 Dynamics and Relaxation Molecular Rotation T1 and T2 relaxation times hemical exhange - kinetics Amide exchange, chemical shift changes Molecular Translation-Diffusion DOSY - Diffusion ordered NMR

110 Dynamics and Relaxation Time scales and molecular motions Atomic fluctuations, vibrations to s <1Å Group motions. (covalently linked units) s < 1 Å 50 Å Molecular rotation, reorientation s Molecular translation, diffusion Rotation of methyl groups s Flips of aromatic rings s Domain motions s Proline isomerization. > 10-3 s hemical exchange (e.g. two protein conformations) Amide exchange Ligand binding

111 Dynamics and Relaxation Time scales and molecular motions Atomic fluctuations, vibrations. Group motions. (covalently linked units) Molecular rotation, reorientation Molecular translation, diffusion Rotation of methyl groups. Flips of aromatic rings. Domain motions. hemical exchange, proline isomerization Amide exchange Ligand binding Influences bond length measurements Relaxation, linewidths, correlation times DOSY NMR 2 NMR 2 NMR 2 NMR hemical shifts 15 N- 1 SQ Transferred NOE measurements

112 NMR as Tool to Study Enzyme motions Entire time range At atomic resolution At equilibrium Under physiologic conditions ps ns μs ms s min hr relaxation (T 1, T 2, het.noe) Relaxation, dispersion (T 1ρ, T 2 ) line shape analysis 2D exchange (NOESY) saturation transfer /D exchange

113 Steady-State State NOE NOE ratio DP WATER