PROTEIN NMR TECHNIQUES PROTEIN NMR TECHNIQUES ///////////// Protein, Proteins, Relaxation, Experiments, Dipolar, Methods, Spectra, Peak, Peaks, Analysis, Techniques, Protein NMR Techniques
Since the advent of NMR, synthetic chemists have had an excellent way to characterize their synthetic products. With the arrival of multidimensional NMR into the realm of analytical techniques, scientists have been able to study larger and more complicated molecules much easier than before, due to the great amount of information 2D and 3D NMR experiments can offer. With 2D NMR, overlapping multiplets and other complex splitting patterns seen in 1D NMR can be easily deciphered, since instead of one frequency domain, two frequency domains are plotted and the couplings are plotted with respect to each other, which makes it easier to determine molecular connectivity.
Spectra are obtained using a specific sequence of radiofrequency (RF) pulses that are administered to the sample, which can vary in the angle at which the pulse is given and/or the number of pulses. Figure 1 shows a schematic diagram for a generic pulse sequence in a 2D NMR experiment. First, a pulse is administered to the sample in what is referred to as the preparation period. This period could be anything from a single pulse to a complex pattern of pulses. The preparation period is followed by a “wait” time (also known as the evolution time), t1, during which no data is observed. The evolution time also can be varied to suit the needs of the specific experiment. A second pulse is administered next during what is known as the mixing period, where the coherence at the end of t1 is converted into an observable signal, which is recorded during the observation time, t2. Figure 2 shows a schematic diagram of how data is converted from the time domain (depicted in the free induction decay, or FID) to a frequency domain. The process of this transformation using Fourier Transform (FT) is the same as it is in 1D NMR, except here, it is done twice (or three times when conducting a 3D NMR experiment).
In 1D NMR, spectra are plotted with frequency (in ppm or Hz, although most commonly ppm) on the horizontal axis and with intensity on the vertical axis. However, in 2D NMR spectra, there are two frequency domains being plotted, each on the vertical and horizontal axes. Intensity, therefore, can be shown as a 3D plot or topographically, much like a contour map, with more contour lines representing greater intensities, as shown in Figure 3a. Since it is difficult to read a spectrum in a 3D plot, all spectra are plotted as contour plots. Furthermore, since resolution in a 2D NMR spectrum is not needed as much as in a 1D spectrum, data acquisition times are often short.
2D NMR is very advantageous for many different applications, though it is mainly used for determining structure and stereochemistry of large molecules such as polymers and biological macromolecules, that usually exhibit higher order splitting effects and have small, overlapping coupling constants between nuclei. Further, some 2D NMR experiments can be used to elucidate the components of a complex mixture. This module aims to describe some of the common two-dimensional NMR experiments used to determine qualitative information about molecular structure.