Structurele Biologie NMR

Transcription

1 MR journey Structurele Biologie MR 5 /3C 3 /65 MR & Structural biology course setup lectures - Sprangers R & Kay LE ature (27) basics of MR (Klaartje ouben: k.houben@uu.nl; 4/2) from peaks to data (ans Wienk; 7/2) from data to structure (Alexandre Bonvin; 9/2) practicals - I. bioinformatics (Gert Folkers; 7/2) II. sequential assignment (Klaartje ouben & Suat Özderikcan; 9-2/2) III. structure determination (ans Wienk & ugo van Ingen; 9-2/2) IV. MR measurement (Karine Loth; 9-2/2) V. analysis protein-protein interaction (Tjerk Wassenaar & Adrien Melquiond; 9-2/2) in groups/caroussel Sugase K, Dyson J, Wright PE ature (27) 2 /65 4 /65

2 course setup reading material - lecture hand-outs (take notes!) - additional, not obligatory! obel lecture by Wuthrich examination - part of written exam, p. II (25 January) on the web - Topics why use MR...? the very basics multidimensional MR MR relaxation & dynamics 5 /65 7 /65 MR basics pros and cons of MR in structural biology

3 Pros... MR is spectroscopy... no need for crystal:! no crystal packing artefacts; solution more native-like potential to study dynamics:! picosecond to seconds time scales, conformational averaging, chemical reactions, folding... easy study of protein-protein, protein-da, protein-ligand interactions Cons not microscopy! MR structure determination is a bit slow... MR structures have a lower quality... MR works best for MW < 5 kda So..! MR and X-ray crystallography are complementary! 9 /65 /65 The protein sample isotope labeling - unlabelled (peptides) - 5 labelled (small proteins < kda) - 5 & 3 C labelled (larger proteins, up to 3-4 kda) - 5, 3 C & 2 labelled (large proteins > 4 kda) very basics of MR protein production (E.coli) - quite a lot & very pure & stable! 5 ul of.5 mm solution -> ~ 5 mg per sample - 3 C labelling is costly, ~k! per sample - preferably low salt, low p, no additives /65

4 uclear spin (rad. T-. s-) 3/65 5/65 uclear spin uclear spin & radiowaves precession m = -! & %E ) & =, hb ) n" = exp($ + = exp( $ + =.9999 n# ' kb T * ' kb T * Larmor frequentie " = #B = $/2% m=! E = µ B! r r E = "µ B = "µz Bz µ = " h I(I + ) I = quantum number µz = " h m m = I, I-, I-2 -I = allowed states!! 4/65 6/65

5 Boltzman distribution Rotating frame m = -! =, hb m =! B n " & = exp $ %E ) & ( + = exp $, hb ) ( + =.9999 n # ' k B T * ' k B T * B " B # = 2! ( " B # = 2! rotating frame: observe with frequency $ 7/65 9/65 et magnetization Chemical shielding n " & = exp $ %E ) & ( + = exp $, hb ) ( + =.9999 n # ' k B T * ' k B T * Local magnetic field influenced by electronic environment 8/65 2/65

6 Chemical shift $= : analogue vs digital Rotating frame: # B ( %! ) 2" #= " B & ( % $ ) 2! #= " B 2! 2/65 FT 25 Signal Fourier Transform Signal Free induction decay = 23/ time (ms) freq. (s-) FT 22/65 24/65

7 Relaxation restoring Boltzmannequilibrium Sensitivity Signal to noise ratio (S/) T2-relaxation - dissappearence of transverse (x,y) magnetization Resolution Line-width /T2 ~ signal line-width 25/65 Relaxation Scalar coupling a.k.a J-coupling T-relaxation 5- - build-up of longitudinal (z) magnetization 27 /65 J! " 26/65 28/65

8 Scalar coupling a.k.a J-coupling Karplus-curve questions 29/65 Key concepts basics of MR nuclear magnetic resonance FT-MR: pulse, rotating frame & chemical shift MR relaxation: T & T2 J-coupling. List at least two reasons why it is more difficult to do MR on very large proteins. 2. ow can the sensitivity of the MR experiment be increased? 3. What factors influence the time it takes for an MR experiment? 4. Is it enough to know all chemical shifts en J-couplings to determine a protein structure by MR? Explain. 5. One could say that we are extremely lucky to be able to do MR, even more so for MRI. Explain. 3/65 32/65

9 multidimensional MR experiments multidimensional MR resolve overlapping signals! enables assignment of all signals encode structural and/or dynamical information! enables structure determination! enables study of dynamics 35/65 2D MR 34/65 36/65

10 3D MR 2D FT t mixing t= t=!t t=2!t t=3!t t=4!t t=5!t t=6!t... F F2 37/65 F2 t F 39/65 nd experiment D single of points encoding information mixing/magnetization transfer t spin-spin interactions 2D t mixing s of points???? 3D x s of points proton A proton B t mixing mixing t3 38/65 4/65

11 encoding information: mixing magnetic dipole interaction (OE) - through space - distance dependent (/r 6 ) - OESY -> distance restraints J-coupling interaction - through 3-4 bonds max. - chemical connectivities - TOCSY,COSY -> assignment - also conformation dependent F "A homonuclear MR OESY t tm A A ("A) A B "A "B ~Å proton A proton B A ("A) (F,F2) = "A,"A B ("B) (F,F2) = "A,"B Diagonal Cross-peak 4/65 F2 43/65 homonuclear MR heteronuclear MR OESY t tm magnetic dipole interaction crosspeak intensity ~/r 6 up to 6 Å COSY TOCSY t t mlev J coupling interaction max. over 3-4 bonds J coupling interaction multiple steps over max. 3-4 bonds 5 - measure frequency of different nuclei; e.g., 5, 3 C - no diagonal peaks - mixing not possible using OE, only via J 42/65 44/65

12 = = J coupling constants - 5 SQC: protein fingerprint Ri Ca C _ = O C Ca Ri+ _ O 45/65 5 structured protein 47/65 heteronuclear MR 5! J-mix block SQC (heteronuclear single quantum coherence)!!! t J-mix block DEC - 5 SQC: protein fingerprint unstructured protein _ Ri Ca C = O C Ca Ri+ _ O J J 5 (" ) (" ) 5 (F,F2) = # 5, # 46/65 48/65

13 !"# $%&'("#)*+,-.%/'.%',*23.% J coupling constants Sequential assigment strips of 3D CA spectrum (5 dimension! to screen) 3Ca (i-) 3Ca (i) (i) 49/65 triple resonance MR t3 J-mix block 5 J-mix block J 5 J Ca(i) 3C 2J Ca(i-) (F,F2,F3) = (# 3 resolve overlapping signals mixing/magnetization transfer DEC OESY, TOCSY, COSY J-mix block t 3C Key concepts multidimensional MR CA J-mix block 5/65 SQC J Ca(i) (" C) JCa(i-) Ca(i), #5(i), #(i)) & (# (" ) 5 3 J ( " ) Ca(i-), #5(i), #(i)) 5/65 triple resonance experiments 52/65

14 questions relaxation & dynamics MR time scales. Sketch the 2D OESY spectrum for a valine in a protein. 2. Sketch the 2D - 5 SQC spectrum for a valine in a protein. 3. Suggest a way to resolve overlap in a 2D TOCSY. protein dynamics protein folding domain motions loop motions side chain motions bond vibrations overall tumbling enzyme catalysis; allosterics fs ps ns s ms s MR R,R 2,OE relaxation dispersion real time MR J-couplings /D exchange RDC 54/65 56/65

15 FAST MOTIO local fluctuating magnetic fields Chapter 5. 7 FAST (ps-ns): rotation correlation time 6 U-factor / Ramachandran Z-score Final Ramachandran plot Z-score U-score -2 B residue number Binduced 2 57/ / Relaxation relaxation time is related to rate of motion R (s - ) FAST (ps-ns): protein 5 flexibility 2 5 R (s-) OE (b) 5 5 R /R residue number residue number 3 R = /T R2 = /T2 8 58/65 C tail loop 6/65

16 MR time scales Key concepts relaxation protein dynamics protein folding domain motions loop motions side chain motions bond vibrations overall tumbling enzyme catalysis; allosterics fs ps ns s ms s time scales fluctuating magnetic fields rotational correlation time (ns) MR R,R 2,OE relaxation dispersion real time MR J-couplings /D exchange RDC fast time scale flexibility (ps-ns) slow time scale (ms-$s): conformational exchange 6/65 63/65 SLOW (#s-ms): conformational exchange A k A B k B summary k ex / #B #A - #B #A frequency (z) frequency (z) SYMMETRICAL EXCAGE equal populations: pa = pb ASYMMETRICAL EXCAGE skewed populations: pa >> pb 62/65

17 basic MR theory where does the MR signal come from? how to generate the MR signal? what information does the MR signal contain? multidimensional MR why use multiple dimensions for protein MR? how to transfer magnetization between spins? protein dynamics which MR parameters give information on fast motions? which MR parameter gives information on slow motions? 65/65 5 / 3 C 66/65 3