Paramagnetic Effects BCMB/CHEM

Transcription

1 Paramagneti Effets BCMB/CHEM 890 0

2 Referenes Expanding the utility of NMR restraints with paramagneti ompounds: Bakground and pratial aspets, Koehler J and Meiler J, Prog. NMR Spet. 59: Paramagneti tagging for protein struture and dynamis analysis, Keizers PM and Ubbink M, Prog. NMR Spet. 58: Exploring sparsely populated states of maromoleules by diamagneti and paramagneti NMR relaxation, Clore GM, Prot. Si. 0: Lanthanide-tagged proteins - an illuminating partnership, Allen KN and Imperiali B, Curr. Opin. Chem. Biol. 4: Protein NMR Using Paramagneti Ions, Otting G, Ann. Rev. Biophys, 39: Paramagneti labelling of proteins and oligonuleotides for NMR, Su X-C and Otting G, J. Biomol. NMR, 46: 0-00

3 Paramagneti Dipolar Spin Relaxation Effets ] 6 3 [ H S H H S H B e o p r SS g R ω ω ω ω ω μ γ π μ = ] [ H S S H H S H B e o p r S S g R ω ω ω ω ω ω μ γ π μ = - = m - e - where m and e are moleular tumbling and eletron spin orrelation times Shortest term dominates, e an add field dependene ω S is large at.7t x0 of 0-9, ω H, terms dominates, R < R

4 Relaxation Enhanement by Free Radials Nitroxides an Identify Interation Sites. Example: Galetin Interating with LaNA

5 Synthesis of a Spin-Labeled N-aetyllatosamine O OH Dhbt-OH O O N N N O HO HO OH O OH HO O OH O ANH NH O. N THF, DCC O. N DMF, DIPEA HO HO OH O OH HO O OH O ANH NH O CH 3 CH 3 CH3 N O. CH 3

6 Intensities in HSQC Experiments are Measures of R transverse proton magnetization during period is major loss H 5 N HI = /4J 5 NS 90-x 80y 90y 90x Battiste and Wagner 000 Biohemistry 39:

7 Change in 5 N HSQC spetrum 800 MHzof Galetin-3 upon addition of LaNa-TEMPO 0 mm 0 mm

8 Distanes from R Equation Residue X-Ray model Å a Spin Label method Å b = 6 ns = 8 ns = 0 ns

9 X-Ray rystal struture of Galetin-3 Seetharamana et al. 998 E84 E65 R86 K7 A45 There are some anomalies ontrol looking at TEMPO alone

10 Proteins Can Also Be Tagged Membrane-Bound myr-yarf-gtp Membrane model: DHPC/DMPC bielles. Complex ~ 70kDA Bielle

11 PREs Give Long-Range Distane Constraints N O r 5 N- H PRE r = 6 N e N m N e N m i= j= r 6 ij

12 PRE Data using H- 5 N HSQC Attenuations S6C-MTSL D67 W66

13 Fitting PREs Required 3-State Averaging for N-terminal Helix Ensemble Averaging: XPLOR-NIH over 3 nitroxide onformers over 3 protein onformers Iwahara,Shwieters,and Clore 004 JACS, 6, Liu Y, Kahn RA and Prestegard JH 00. Nature Strut Mol Biol. 7:876-8.

14 Myr-yARF-GTP FAPP-PH Interations

15 FAPP paramagneti perturbations from T56C ARF E80 A78 80 G67 W5 N58

16 FAPP ARF doked models using PREs 80 Membrane 90

17 Metal Ions Have useful Paramagneti Properties Bertini, Luhinat, Parigi, 00 Solution NMR of Paramagneti Moleules

18 Short Eletron Spin Lifetimes: Contat Shifts, Pseudo-Contat Shifts and Field Alignment Bertini, 00

19 Curie Spin Relaxation Still Ours for Short e 3 4 B r H o 6 r H r r B B J o PRE T k J J g ω μ γ π μ λ = Comes from exess population of β spin state there is a net average magneti moment Note field squared dependene lose signals at 0Å at 8T Best to use an ion with low J

20 Identifying a Dimer Interfae with Gd-DTPA Lee H-W, Wylie G, Bansal S; et al. Prot. Si. 9: Comparison of rosspeak attenuation at high and low protein onentration. Ratio of effets givens protetion fator. Red are proteted

21 Pseudo Contat Shifts and Moleular Alignment Bertini, I., et al. 00. Conepts in Magneti Resonane 4: PCS = πr 3 [ Δχ ax 3os θ Δχ rh sin θ os ϕ] RDC = 0π B o γ r H 3 NH γ k N B hs T [ Δχ ax 3os Θ Δχ rh sin Θos Φ] These effets depend on anisotropi suseptibilities Size of Indued dipoles depends on moleular orientation Hene, rotational averaging does not redue dipolar field to zero in PCS ase Differene in interation with B 0 for different orientations results in field indued alignment and measureable RDCs

22 Metal binding peptide sequenes EF-hand an be added to protein onstruts. Pseudo-ontat shifts and RDCs provide validation of struture and positioning of ligands Lanthanide Tagged Galetin-3 Zhuang T, Lee H-S, Imperiali B et al., Prot. Si. 7:

23 PCSs of Galetin-3-LBT Lu 3 -Blak Dy 3 -Green

24 5 N- H RDCs and PCSs of Galetin-3-LBTDy 3 Agreement with rystal struture A3K Agreement of RDCs and PCSs suggest a rigid model an be used

25 PCSs for.5mm Latose with 0.5mM Galetin-3 at H frequeny 800MHz PCS obs Hz PCS bound Hz Bak-alHz Glα H -0± -48±5-45 H -6± -53±5-55 H3-8± -4±5-46 H5-0± -5±5-53 Gal H -0±3-50±5-49 H -9±3 --45±5-49 H3-9±3 45±5-43 H4 -±3-55±5-5 H5 -±3-55±5-59