Lecture #7 (2D NMR) Utility of Resonance Assignments

Transcription

1 Lecture #7 (2D NMR) Basics of multidimensional NMR (2D NMR) 2D NOESY, COSY and TOCSY 2/23/15 Utility of Resonance Assignments Resonance Assignments: Assignment of frequency positions of resonances (peaks) in the NMR spectrum to specific atoms in the macromolecule. Macromolecule H Protein folding Resonance Assignments Dynamics (10-12 to seconds time scale) 1D 1H NMR spectrum Protein pka measurements Mapping ligand binding surfaces (drug discovery) Multidimensional experiments required to resolve peak overlap Structure 1

2 COSY: COrrelation SpectroscopY 2

3 Basic 2D COSY COSY: COrrelation SpectroscopY Review: the effect of 90 o pulses applied on different axes Before After o 90 o x pulse (B1) B 1 Bo Bo Mo Mo z x Detected component The amount of component in transverse plane is Mo sin (360* *t1) (Blue). Only this component is detected. Similar to 1D it will rotate in the transverse (xy) plane at a frequency 3

4 Peak Peak height proportional to: Mo sin (360 t 1 ) z F2 frequency domain x t 1 = 0 sec interferogram intensity Magnitude of peak maxima oscillates at sin (360* a*t 1 ) 4

5 Amplitude of peak is modulated by Cos (2 * a *t 1 ) Interferogram (the 2 nd ) a (direct dimension) a a 5

6 Case II (transfer during mixing period) t1 (F1) t2 (F2) Key experimental parameters for multidimensional NMR experiments (1) Sweep-width in indirect dimension: Controlled by the amount the t1 delay is incremented between successive experiments. (t 1 ): Amount that the t1 delay is incremented between successive experiments. SW(F1) = 1/( (t 1 )) ***Note: it s more complicated than this!! It depends upon how the experiment was performed. There are many ways to obtain quadrature detection in the indirect dimension (different ways to sample the signal so as to discern positive and negative frequencies in the indirect dimension). In these different methods (STATES, STATES-TPPI, TPPI, ECHO-ANTIECHO) the delay is not always incremented on each successive t1 increment! (t 1 )= 100 s t 1 =0 s Example: (t1)= 100 s 1 / (100 s) = 10,000Hz t 1 =100 s t 1 =200 s t 1 =300 s SW(F1) = 10,000Hz F2 (direct dimension) F1 (indirect dimension) t 1 =400 s (t 1 )= 50 s t 1 =0 s Example: (t1)= 50 s 1 / (50 s) = 20,000Hz t 1 =50 s t 1 =100 s t 1 =150 s SW(F1) = 20,000Hz F1 (indirect dimension) t 1 =200 s F2 (direct dimension) 6

7 (2) Digital resolution (DR) in indirect dimension Interferogram The more t 1 increments you collect the better the digital resolution in the indirect dimension. interferogram (t 1 ) 128 t1 increments 512 t1 increments Nyquist theorem: To properly determine the frequency of an oscillating signal must sample it at least twice per wavelength Typically 256 to 1024 experiments (increments) are recorded depending on the desired digital resolution in indirect dimension 7

8 (2) Digital resolution (DR) in indirect dimension A Indirect dimension (F2) Indirect dimension (F2) B Example: SW(F1) = 10,000 Hz expts. recorded = 128 DR= Hz/point 128 1D files (serial files) collected in t1 dimension Example: SW(F1) = 10,000 Hz expts. recorded = 512 DR = 39.1 Hz/point 512 1D files (serial files) collected in t1 dimension Mid-80s Early 90s late 90s - to present Different strategies to assign proteins Homonuclear Methods (1H 2D COSY, TOCSY etc.) Proteins < kd (< ~100 aa) Old School Triple Resonance (3D/4D 1H, 13C & 15N) (most common) Quadruple Resonance (3D/4D 1H, 13C & 15N) Proteins < 30 kd (< 250 aa) Proteins < 85 kd (< 723aa) JACS (2002) 124: Direct dimension (F2) 8

9 Important 2D experiments used in homonuclear assignment methods 2D COSY 2D NOESY 2D COSY 2D TOCSY NOESY (Nuclear Overhauser Enhancement SpectroscopY): Connects hydrogen atom that are separated by < 5 Angstroms (<0.5nanometers) TOCSY (TOtal Correlation SpectroscopY): Connects hydrogen atoms that are part of the same spin network (spin system). Some people refer to this as a HOHAHA experiment. COSY (COrrelation SpectroscopY): Connects hydrogen atoms that are separated by three bonds or less *Homonuclear: Frequency of single atom type (hydrogen in these experiments) is observed in frequency dimensions In the COSY experiment, magnetization is transferred by scalar coupling. Protons that are more than three chemical bonds apart give no cross signal because the 4 J coupling constants are close to 0. Therefore, only signals of protons which are two or three bonds apart are visible in a COSY spectrum (red signals). The cross signals between HN and Halpha protons are of special importance because the phi torsion angle of the protein backbone can be derived from the 3 J coupling constant between them. 9

10 2D TOCSY 2D NOESY In the TOCSY experiment, magnetization is dispersed over a complete spin-system (set of hydrogen atoms) of an amino acid by successive scalar coupling. The TOCSY experiment correlates all protons of a spin system. Therefore, not only the red signals are visible (which also appear in a COSY spectrum) but also additional signals (green) which originate from the interaction of all protons of a spin system that are not directly connected via three chemical bonds. In the NOESY experiment, magnetization is transferred between 1H nuclei when they are < ~5A apart The closer the nuclei the stronger the cross-peak (proportional r -6 ) 10

11 Example 2D NOESY spectrum 1 mm Protein dissolved in water (55.5M H2O) 2D NOESY Experiment (transfer of magnetization via dipole-dipole interactions) H I 1D spectrum <6 angstroms H S < 5A Ha Hb (f) (g) < 5A Hc (a) (b) (c) (d) (e) I S 0 Hz frequency Example of single increment in the experiment (a) IS (b) (c) I cos (360* S *t 1 ) S I S Hb Hc Ha F2 F1 F2 (ppm) F1 (ppm) (d) I S m period (e) I S Non-equilibrium condition. Populations of S-spin inverted Transient NOE condition. Spin S has been inverted, while spin I has not. Hc Hb F2 Ha F1 NOE effect causes intensity of I to be reduced (negative NOE). The amount of S on z axis depends on cos (360* S *t 1 ). ( its projection onto the Y axis at step (c)) Therefore the intensity reduction in I is modulated by cos (360* S *t 1 ). Wo Assuming Negative NOE (Wo dominates) 11

12 2D NOESY Experiment (transfer of magnetization via dipole-dipole interactions) (f) H I 1D spectrum <6 angstroms H S Intensity of cross-peak related to mixing time (t m ) (a) (b) (c) (d) (e) I S 0 Hz frequency (d) (f) I S m period (e) I S Intensity of I modulated by amount of S magnetization at beginning of mixing time that is inverted. This in turn depends upon larmor frequency of spin S in t1 dimension. Therefore frequency of S nucleus in F1 dimension has been encoded within the amplitude of spin I when it is detected Rate of build-up of cross-peak is inversely proportional to distance separation S Direct detection of I oscillating at frequency I I Rate of NOE build-up (t2 -> F2) (t1 -> F1) I F2 (direct dimension) S F1 (indirect dimension) ms Typical Mixing time We record NOESY spectra at a fixed tm value where intensity is proportional in inter-hydrogen atom separation (r IS ) 12

13 The sign of the cross-peak in NOESY depends on the molecular tumbling time (size) of the molecule or whether it is caused by chemical exchange. Large molecule (- NOE) Biomolecules! Wo dominates I F2 (direct dimension) S F1 (indirect dimension) Small molecule ( NOE) - I F2 (direct dimension) - H I S F1 (indirect dimension) r IS H S Cross-peaks from NOEs Protein requirements for homonuclear resonance assignment methods (1) Protein molecular weight < kds (<~100aa) homonuclear assignment methods don t work for larger proteins because: (a) Increased spectral complexity. (signal overlap) (b) Increased proton transverse relaxation (smaller T 2 values cause proton magnetization to decay too rapidly. Important homonuclear proton COSY and TOCSY experiments no longer work ) (2) protein concentration > 0.25 to 0.5mM. Data collection for less concentrated proteins takes too long. S/N is proportional to Sqrt (# of scans). 16 times as many scans need to be acquired on a 0.125mM sample to get the same S/N ratio as obtained when the same experiment is acquired on the same sample concentrated to 0.5mM. (3) Salt (NaCl or KCl) concentrations <500mM Higher salt concentrations degrade the performance of the NMR probe reducing signal to noise. (4) Must not significantly aggregate. Aggregation increases apparent c. This decreases the T2 values. Cross-peaks from chemical exchange a Chemical exchange: During the mixing time ( m) the magnetic environment of hydrogen atom changes (chemical shift changes) as a result of conformational rearrangement or chemical reaction b F1 (indirect dimension) e.g. a b Conformational change (5) dissolved in non-protonated solvents. Protonated solvents give rise to signals in the NMR spectra that mask those of the protein. Note: Most experiments are performed on samples containing water which gives rise to a water peak at ~4.76ppm. This huge signal is normally eliminated by using water suppression techniques during the experiment (e.g. selective excitation, solvent presaturation, magnetic field gradients) Some good NMR buffers: (a) 50mM PO 4 /100mM NaCl/7% 2 H 2 O (D 2 O) (b) 50mM Tris-d11/100mM NaCl/7% 2 H 2 O (D 2 O) (c) deuterated acetate/100mm NaCl/7% 2 H 2 O (D 2 O) Frequently 0.01% Sodium Azide is also added to prevent microbial growth F2 (di t di i ) 13

14 Why do we need 2 H 2 O in the solvent? The magnetic field generated by the superconducting magnet changes with time ( drifts ) 2 H 2 O is used to lock the magnetic field (keep it from changing) during data acquisition On a 500 MHz NMR magnet 1 H frequency = 500 MHz 2 H frequency = 76.8 MHz (6) Sample should be soluble at ph values < 8.0 At alkaline ph values have increased rate of exchange of protein hydrogen atoms with solvent. This tends to attenuate the signals arising from exchangeable atoms. Solvent Exchange H A N H B O H C k intr H B N H A O acid or base catalyzed H C H B H A N H B (-) (-) O O N H C H A H C Example of base catalyzed NMR Experiment Lock System H A N Observed chem. shift of H A ~ chem. shift of H 2 O H 2 O 1H RF excitation & detection (500 MHz) 2 H RF excitation & detection (76.8MHz) Computer (signal processing and storage) Automatically adjust magnetic field to counter-act field drift Monitor 2 H Signal position Chemical shift of atom H A = P H A N Fraction of N-H A ~10-3 M A P O H A H H 2 O B ~ H 2 O Fraction of H A -O-H B ~55 M 14

15 Protons that exchange H N Backbone amide Trp indole His sidechain Sample manipulation trick: Change buffer into D 2 O solvent to simplify the NMR spectra H H O H H O H H O O H Ser/Thr/Tyr Arginine sidechain N-term residue, Lys sidechain O C.. S H.. HO Carboxylic acid (C-term residue, Asp, Glu) Asn, Gln sidechain Cys sidechain N-term.. N C C N C C N C C.. CH CH HO CH 3 H 3 C CH 3 Buffer exchange protein into 100% D 2 O solvent (cycle to higher ph to facilitate exchange: ph <7 ph 8 ph <7 ) H C-term water sample 50mM PO 4 100mM NaCl 7% 2 H 2 O (D 2 O) 0.01% NaN 3 Not observable in NMR spectrum D H O D H O D H O N-term.. N C C N C C N C C.. C-term CH CH DO CH 3 H 3 C CH 3 H D2O sample 50mM PO 4 100mM NaCl 99.99% 2 H 2 O (D 2 O) 0.01% NaN 3 15