Observaciones y datos obtenidos en OAGH

The problem o f signal overlap in the N M R spectra o f biomolecules can be relieved to a considerable extent by the use o f 2-D N M R experiments. The developm ent o f 2-D N M R techniques was a vital breakthrough in the use o f N M R in m olecular biology. T he main features o f m ulti-dim ensional N M R can be explained by considering an N M R experim ent in term s o f its principle com ponents.

A simple 1-D N M R experim ent (a pulse-and-collect experiment) consists o f a radio-frequency pulse causing nuclear excitation, evolution o f the magnetisation and detection o f the N M R signal (the FID ) in the tim e-dom ain. Fourier transform ation o f the F ID yields a 1-D spectrum in which intensity peaks are plotted along a frequency axis {Fignre 2-3). A typical 2-D NM R experim ent is only slightly m ore complicated. In order to collect data with a second frequency dim ension, it is necessary to introduce a second time elem ent into the N M R experim ent. This is essentially achieved by perform ing two consecutive pulse-and-collect experim ents b ut w ithout allowing

Figure 2-3: A schematic outline of the basic 1-D and 2-D N A I R experiments.

1 -D NMR Fourier transform 1-D spectrum Frequency 2 - D NMR 2-D sp ectrum 90° *’ 90° *2 Fourier g. transform ® ( x 2 ) u- Frequency

therm al equilibrium to be reached betw een the experim ents. This com prises the fundam ental concept o f a 2-D N M R experiment: the probing o f a spin-system which is in a non-equilibrium state {Figure 2-3).

The typical 2-D N M R experim ent can be thought o f as containing four sections: Preparation — Evolution ( t j - Mixing (x ^ — D etection (t?). T he preparation section involves pulsing the system such that the m agnetisation reaches the desired non-equilibrium state, this preparation varies depending on the type o f experim ent. T he nuclear spins are allowed to ‘evolve’ for a time t^ during the evolution section. D uring t^ evolution, the m agnetisation is ‘labelled’ with the L arm or frequencies o f the specific nuclear spins excited during the preparation section. Thus, during the evolution time t,, a nucleus A may precess with its characteristic frequency. D uring the mixing time (X^J, if a second nucleus B is coupled to nucleus A it may interact through a ‘coherence transfer’ pathway, such that B will becom e excited. The frequency o f nucleus B will be subsequently detected in the F ID collected during t,. In this way, the m agnetisation is ‘labelled’ with com ponents com ing from nucleus A and nucleus B, m odulated as a function o f t^.

A fter each pulse sequence an FID is collected, com prising a signal o f intensit)' which varies with time. T he ‘second dim ension’ o f a 2-D experim ent is introduced by collecting a series o f the four-step experim ents described which differ only according to a successive increm entation o f their time delays in the t, evolution period. In this way, detection in t, followed by Fourier transform ation produces a signal from nucleus B which is m odulated with the frequency nucleus A, and in the 2- D frequency spectrum produced there will exist a ‘cross-peak’ at the intersection o f

Figiin 2-4: The results of the 1 -D N M R experiments with different settings form a 2-D matrix. Fourier transforms along /; and yield signals with two frequency components in a 2-D contour map. Cmss-peaks indicate that nuclei A and B are pin-coipled.

Evolution Mixing Detection

2-D spectrum to 90^ 90^ 90^ transform ® ( x 2 )

À

B

_o

A

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A

B

the two nuclear signals {Figure 2-4). Most 3-D and 4-D NMR experiments are essentially built from blocks o f 2-D experiments which can be added after the experimental ‘evolution — mixing period’ block, prior to the detection period.

The process o f coherence transfer, mentioned in the outline o f a 2-D NMR experiment above, cannot readily be described using the model o f the classical formalism. The more powerful product operator formalism is required in order to explain the mechanisms by which phase coherence allows magnetisation transfer between coupled nuclei during an NMR experiment. A full description o f this process is beyond the scope o f this thesis, but it can be noted that the principle differences between 2-D experiments are contained within the pulse schemes during the ‘Mixing’ period outlined in the typical 2-D NMR experiment given earlier. It can be shown that the spin coupling process gives rise to cross-peaks in the 2-D spectrum which occur at the intersection o f the chemical shifts corresponding to the coupled nuclei. Spin coupling can either be a scalar coupling occurring through bonds, or a dipolar coupling reflecting through-space interactions. The result o f this is that there are two different types o f basic 2-D NMR experiments which have been designed in order to investigate the different couplings. Correlated spectroscopy (COSY) provides the basis for measuring through-bond couplings, such that in a COSY spectrum cross­ peak resonances occur for chemically bonded nuclei. Many variations o f this experiment have been designed in order to gain information which allows the assignment o f resonance patterns to specific amino acid types. In particular, the TOCSY (total correlation spectroscopy) experiment is used to correlate scalar- coupled protons within a spin-system. TOCSY experiments yield data describing long range through-bond connectivities; however, the coupling detected does not efficiently traverse the peptide bond such that TOCSY experiments can only provide intra-residue information.

The second type o f NMR experiment is described as a nuclear Overhauser effect spectroscopy (NOESY) experiment. The nuclear Overhauser effect describes the change in intensity o f a resonance when a neighbouring nucleus is selectively irradiated. In a NOESY spectrum, cross-peaks occur between resonances which are connected by a through-space dipolar coupling. The intensity o f the cross-peak is proportional to the inverse sixth power o f the intemuclear separation, with the practical result that NOESY cross-peaks are not usually observed for nuclei separated by greater than 0.5 nm. NOESY experiments therefore allow the collection o f data

which gives information on the 3-D organisation o f the nuclei within the sample, i.e. nuclei close in space can be identified. Firstly, this information allows the amino acid resonance types identified by COSY experiments to be recognised in terms o f their position in the primary sequence o f the protein. This essential procedure is known as the ‘sequential assignment strategy’ (Wagner and Wiithrich, 1982). In addition, the N OESY experiments provide spatial information concerning the intemuclear distances o f atoms in amino acids which may be distant in the primary protein sequence but close in 3-D space. Together, the ability o f the COSY-type experiment to allow assignment o f the spectral resonances plus the spatial information provided by NOESY experiments forms the basis o f the methodology for a structural determination by NMR. A more detailed description o f the procedure for biomolecular structure determination is not required here, but can be found in the literature (Wiithrich, 1986).