Secondary protein structure techniques

NMR has become an indispensable analytical technique for studying rates of H D exchange at specific sites when a protein is deuterated. Such exchange rates provide information on secondary protein structure and are useful in investigating the mechanism of protein folding. 

Ihe rule-based approach to protein structure prediction is obviously very reliant on th quality of the initial secondary structure prediction, which may not be particularly accurate The method tends to work best if it is known to which structural class the protein belongs this can sometimes be deduced from experimental techniques such as circular dichroism... 

The secondary structure elements, formed in this way and held together by the hydrophobic core, provide a rigid and stable framework. They exhibit relatively little flexibility with respect to each other, and they are the best-defined parts of protein structures determined by both x-ray and NMR techniques. Functional groups of the protein are attached to this framework, either directly by their side chains or, more frequently, in loop regions that connect sequentially adjacent secondary structure elements. We will now have a closer look at these structural elements. 

The complexity of quality control for proteins, as compared to small molecules, is most evident in the requirements for proof of structure. Many small molecules can be fully characterized using a few spectroscopic techniques (e.g., NMR, IR, mass spectrometry, and UV) in conjunction with an elemental analysis. However, proving the proper structure for a protein is much more complex because 1) the aforementioned spectroscopic techniques do not provide definitive structural data for proteins, and 2) protein structure includes not only molecular composition (primary structure) but additionally, secondary, tertiary, and, in some cases, quaternary features. Clearly, no single analytical test will address all of these structural aspects hence a large battery of tests is required. 

The secondary structure of proteins may also be assessed using vibrational spectroscopy, fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy both provide information on the secondary structure of proteins. The bulk of the literature using vibrational spectroscopy to study protein structure has involved the use of FTIR. Water produces vibrational bands that interfere with the bands associated with proteins. For this reason, most of the FTIR literature focuses on the use of this technique to assess structure in the solid state or in the presence of non-aqueous environments. Recently, differential FTIR has been used in which a water background is subtracted from the FTIR spectrum. This workaround is limited to solutions containing relatively high protein concentrations. 

In another variant on the use of paramagnetic species to probe protein structure, Zhao et al.2m determined the secondary structural features of A8-3-ketosteroid isomerase using 3D NMR techniques on the l5N,l3C-labelled protein. This enzyme catalyses the conversion of A5- to A4-3-ketosteroids and is a homodimer of 125 amino acids per subunit. The NMR studies were undertaken on the steroid-bound protein. The amino acids near to the steroid were confirmed by binding a steroid incorporating a spin label and monitoring the disappearance from the 15N- H HSQC spectrum of the cross-peaks associated with these residues. 

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