Disordered proteins characterization methods

Number of Proteins and Residues in Databases of Intrinsically Disordered Protein Characterized by Various Methods... 

To obtain statistically significant comparisons of ordered and disordered sequences, much larger datasets were needed. To this end, disordered regions of proteins or wholly disordered proteins were identified by literature searches to find examples with structural characterizations that employed one or more of the following methods (1) X-ray crystallography, where absence of coordinates indicates a region of disorder (2) nuclear magnetic resonance (NMR), where several different features of the NMR spectra have been used to identify disorder and (3) circular dichroism (CD) spectroscopy, where whole-protein disorder is identified by a random coil-type CD spectrum. 

Three groups of disordered proteins have been assembled, with the groups defined by the experimental method used to characterize the lack of ordered structure. Because the focus has been on long regions of disorder, an identified disordered protein or region was not included in these groups if it failed to contain 40 or more consecutive residues. Disordered regions from otherwise ordered proteins as well as wholly disordered proteins were identified. Table I summarizes the collection of sequences in this database. 

Although the disordered proteins were characterized by three different methods, all three datasets of disordered amino acids showed semi-quantitatively similar changes for 16 of the 20 amino acids (Fig. 1, top). Because the three methods rely on completely different underlying biophysical principles for determining disorder, substantial compositional differences among the datasets were expected surprisingly, however, such differences were not observed. Thus, the compositions of the disordered proteins (Fig. 1, top) likely indicate inherent tendencies of this type of protein. 

Although the characterization of the 8-form by rotatory dispersion requires further work, the information that is available indicates that it can be distinguished by its rotatory properties from pure forms of other conformations, helices of both senses and disordered chains, in solution. The capacity of the method to assess mixtures of this conformation with helices and disordered regions, as may occur in certain proteins, will be given further consideration in Section IV, G. 

We have also begun to structurally characterize fibrillar states of a-synuclein (140 residues) and of the K19 domain of protein tau. Both systems are involved in possibly related neuro-degenerative diseases.Preliminary results of two- and three-dimensional correlation spectroscopy, as outlined above, reveal that many of the NMR detectable resonances exhibit narrow or line widths, consistent with the occurrence of well-ordered domains in both proteins. On the other hand, other segments of the considered proteins cannot readily be detected suggesting that they may display static or dynamic disorder under the experimental conditions considered. While the effect of molecular dynamics of NMR relaxation parameters has been studied in the context of solution- or solid-state NMR for a long time, recent progress in the study of uniformly labelled proteins by MAS NMR has created novel means by which to study dynamics of entire polypeptide sequences using 2D methods. For example, Bockmann and Emsley have... 

Because domains can be considered independent structural and functional units, each domain can be analyzed independently once it has been determined that the query protein contains more than one domain. The identification of functional domains can be performed directly by matching the entire query sequence or a portion of it to a profile from a domain database. Alternatively, the existence of functional domains can be evaluated through indirect inference. For instance, if the query protein contains a well-characterized domain that matches a database profile and the rest of the sequence is not covered by any known domain, that uncovered region (provided it has a reasonable length) can be assumed to contain an additional domain. For cases in which there are no matches to domains or protein families in databases, the existence of multiple domains in the protein of interest can still be inferred through other methods. For example, the connectors between domains tend to be disordered or flexible linkers. Accordingly, predictions of disorder or composition bias, linker predictions, or secondary-structure predictions can be used to infer the spatial location of uncharacterized domains. 

Using the above described methods, Christodoulou and coworkers report in-cell NMR characterization of the secondary structure populations of a disordered conformation of a-Synuclein within E. coli Cells. The small protein a-Synuclein populates a highly dynamic state that has the same characteristics as the disordered monomeric form observed in aqueous solution studied by them previously using MD simulations. 

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