Secondary Structures of Proteins: A Comparison of Models and Experimental Results

Abstract

Secondary structure predictions of proteins were compared to experimental results by wide-line 1 H NMR. IUPred2A was used to generate predictions of disordered protein or binding regions. Thymosin-β 4 and the stabilin-2 cytoplasmic domain were found to be mainly disordered, in agreement with the experimental results. α-Synuclein variants were predicted to be disordered, as in the experiments, but the A53T mutant showed less predicted disorder, in contrast with the wide-line 1 H NMR result. A disordered binding site was found for thymosin-β 4 , whereas the stabilin-2 cytoplasmic domain was indicated as such in its entire length. The last third of the α-synuclein variant’s sequence was a disordered binding site. Thymosin-β 4 and the stabilin-2 cytoplasmic domain contained only coils and helices according to five secondary structure prediction methods (SPIDER3-SPOT-1D, PSRSM, MUFold-SSW, Porter 5, and RaptorX). β-Sheets are present in α-synucleins, and they extend to more amino acid residues in the A53T mutant according to the predictions. The latter is verified by experiments. The comparison of the predictions with the experiments suggests that helical parts are buried.

Wide-Line NMR Melting Diagrams, Their Thermodynamic Interpretation, and Secondary Structure Predictions for A30P and E46K α-Synuclein

“…This method predicts almost no β-sheets in the WT α-S but a markedly increased β-sheet content for the two mutants (Figure A). On the contrary, the eight-state predictions forecast the largest β-sheet content for WT α-S, which is in accordance with our earlier secondary structure calculations …”

“…The MDs of A30P and E46K α-S ( Figure 1 ) are characteristic of IDPs. They show the first constant amount of mobile hydration water at relatively high temperature/functional normalized temperature or potential barrier 19 ( T fn0 , t 0 , or E a,0 , Table 1 ) compared to globular proteins (see the Supporting Information of ref ( 17 )), and they have an intensely elevating section from E a,1 on, in contrast to the constant number of mobile water molecules per amino acid residue, naa (see Materials and Methods ) values of globular proteins in the same temperature range.…”

“…On the contrary, the eight-state predictions forecast the largest β-sheet content for WT α-S, which is in accordance with our earlier secondary structure calculations. 17 …”

“…Protein preparation and wide-line NMR measurements were described in former publications (ref ( 16 ) and the Supporting Information in ref ( 17 )). The applied three-state SS prediction methods are Brewery, Jpred4, Porter 5.0, PSIPRED, PSRSM, RaptorX, SCRATCH, and SPIDER3.…”

“…Here, our previous work on WT and A53T α-S was completed with new results on two other familial mutants, A30P and E46K α-S. We measured the melting diagrams (MDs, relative ratios of mobile water as a function of normalized functional temperature; see the Supporting Information of ref ( 17 )) by wide-line NMR, i.e ., the amount of mobile hydration water as a function of temperature, to get information on the steps and gradience in the development of full hydration of α-S. Secondary structure (SS) predictions on the same proteins were calculated and compared to the experimental results by wide-line NMR.…”