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Metodi di indagine di molecole biologiche 2008

Metodi di indagine di molecole biologiche 2008. Piccioli-Turano.

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Metodi di indagine di molecole biologiche 2008

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  1. Metodi di indagine di molecole biologiche2008 Piccioli-Turano Spettroscopia NMR di molecole Biologiche.Aspetti fondamentali della tecnica (Recupero nozioni di base). Esperimenti multidimensionali. NMR eteronucleare. Risonanza Tripla. Assegnamento. Indice del Chemical Shift. Parametri NMR per la determinazione strutturale di molecole biologiche (nozioni di base). Dinamica Interna.Accessibilità al solvente. Idratazione. Interazioni. Sistemi ad alto peso molecolare.

  2. Metodi di indagine di molecole biologiche2008 Piccioli-Turano Laboratorio Lab 1. Lo spettrometro NMR: Set up di un esperimento. Rudimenti. Hardware e configurazioni Lab2. Acquisizione di esperimenti multidimensionali omonucleari ed eteronucleari (tutoriale). Lab3. SOD. Acquisizione spettro HSQC, NOESY ed HSQC-NOESY Lab 4. Processing ed analisi spettri acquisiti (data station) Lab 5. Tripla risonanza. Acquisizione spettro 3D, HNCA, HNCO. Lab 6 Analisi degli spettri. Lab 7. R1 ed R2. Acquisizione di spettri Lab 8 Fitting ed interpretazione Lab 9 Metabolomica Lab 10. TROSY e CRINPET

  3. Basics Concepts • Nuclear spins • Magnetic field B0 • Energy • Sensitivity • The NMR transition • Larmor Frequency • Magnetic field B1 • The rotating frame • A pulse!

  4. Nuclei NMR-attivi Tutti i nuclidi con un numero quantico di spin nucleare I diverso da zero sono NMR attivi, Ovvero possono essere studiati via NMR I=1/2 11H, 136C, 3115P, 199F, 157N I=1 21H, 147N I=3/2 2311Na3517Cl I=5/2 178O 2713Al

  5. Magnetic moment I= spin quantum number m= Magnetic quantum number (-I…+I) I2 = (h/2p)2[I(I+1)] Spin magnetic moment m=gI In the presence of a magnetic field B0 Iz=(h/2p)m Spin angular momentum mz=gIz =g(h/2p)mz(-g(h/2p)I…. g(h/2p)I)

  6. Many atomic nuclei possess a small magnetic dipole (nuclear magnetic moment) , in the same direction as the angular momentum vector I. In an external magnetic field B0, the nuclear magnetic moment m is limited by quantum mechanics to a distinct set of orientations or spin states related to the spin quantum number I. This interaction of nuclear magnetic moment with B0 is called the Zeeman interaction. For spin ½ nuclei, the two possible orientations are Nuclear Magnetic Resonance I m m =-1/2 m =1/2 mz=gIz =g(h/2p)mz

  7. E= -m•B0 The energy of the NMR Transition DE=g(h/2p)B0  NUCLEUS • Sensitivity m=-1/2 DE=h0 E m=+1/2 B0 MAGNETIC FIELD B0 Larmor Frequency The two Zeeman level are degenerate at B0=0

  8. Different Isotopes Absorb at Different Frequencies 15N 2H 13C 31P 19F 1H 50 MHz 77 MHz 125 MHz 200 MHz 470 MHz 500 MHz low frequency high frequency

  9. Nuclear Magnetic Resonance • The two orientations for spin ½ nuclei such as 1H or 13C have different energy level and therefore slightly different populations at equilibrium. The slightly excess of spins in the +1/2 state is called the Boltzman distribution and is proportional to the square of the magnetic moment m, or (gB02) • Transitions between states occur with the absorption or emission of a quantum of energy from an electromagnetgic field at precisely the energy difference between the states hn (the Larmor frequency).

  10. The sensitivity of NMR transition:Boltzmann distribution DE=ghB0/2p Nup/Nlow=e-DE/kT B0=9.4 T; T=300 K, 1Hg= 2.675x108 rad T-1 s-1 DE=2.65x10-25 J kT= 4.14x10 -21 J . . (Nlow-Nup)/(Nlow+Nup) =DE/2kT DE/kT=10-4 1:31000 DP/P

  11. dm/dt=m(t)^B0(t)

  12. Mz=-1/2 hg/2p Mz=1/2 hg/2p w0=2pn0 w0=gB0 n0=gB0/2p E=-mB0 = -ghmzB0/2p DE=ghB0/2p STILL, NO NMR EXPERIMENT

  13. M0 B0 M0 = Sm = Mz Mx = My = 0 I=1/2 E = -mB0 E = -gmB0 a: m = +½ ma = +½g Ea = -½gB0 DE = gB0 = w0 = hn0 b: m = -½ mb = -½g Eb = +½gB0

  14. M0 = Sm = Mz DE = gB0 = w0 = hn0

  15. A pulse! B1 is a radiofrequency transmitter B1(t)= B1  cos w1t

  16. A pulse! Double precession dM(t)/dt= M(t)^B(t) B(t)= B0 + B1(t) dM(t)/dt= M(t)^B0 + M(t)^B1 (t) = M(t)^B0 + M(t)^|B1| cosw1t (t) Precession around B1 (axis defoned in the xyplane and rotating at speed w1) Precession around B0 (z axis)

  17. Laboratory Frame • Nuclear frequency n1= precession frequency of magnetic field B1 A pulse!

  18. The Rotating frame • X’,y’,z’ =laboratory frame • X,y,z,=rotating frame (rotating at the frequency n1) In the rotating frame,there is no frequency precession for m and the radifrequency B1 is seen as a static magnetic field The static magnetic field B0 is not observed in the rotating frame

  19. Laboratory Frame Fly A (Laboratory Frame) jam The movement of Fly B as seen by Fly A Fly B Rotating Frame jam Fly B The movement of Fly B as seen by Fly A Fly A (Rotating frame)

  20. Precession in the laboratory frame dM/dt=M^B dM/dt=M^(B-w0/g) If B = B0 dM/dt=0 R.F. at freq. w0 L.F. If B = B0+B1 dM/dt=M^(B0 +B1 -w0/g) Rotation! dM/dt=M^B1 = w1g Dw0 If ww0 B1 Dw0 dM/dt=M^(B+B1 -w0/g) dM/dt=M^(B1+(w-w0/g)) dM/dt=M^(B1+(Dw/g))

  21. Precession in the laboratory frame Mxy from any nuclear spin not exactly on resonance, will also precess in the x’y’ plane at the difference frequency Dw0. Dw0

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