Comparison of residual alpha- and beta-structures between two intrinsically disordered proteins by using NMR.

Intrinsically disordered proteins contain some residual structures, which may fold further upon binding to the partner protein for function. The residual structures observed in two intrinsically disordered proteins, including the C-terminal segment of peripherin-2 (63 residues) and measles virus nucleocapsid protein Ntail (125 residues), were compared using NMR. Differences in the chemical shifts of alpha-, beta- and carbonyl carbons between the observed structure and calculated random coil revealed the existence of a helix and some possible beta-structures in both proteins. The intensity of signals in the C-terminal segment of peripherin-2 in NMR spectra was informative and locally low, particularly in the middle and N-terminal parts: this suggested the broadening of the signals caused by the formation of residual structures in those areas. Furthermore, the protection of exchange of amide protons was significantly observed at the N-terminus. Conversely, the intensities of signals for Ntail were random beyond the overall areas of protein, and indicated no characteristic pattern. Only a faint protection of amide-proton exchange in Ntail was observed in the C-terminus. It was concluded that Ntail was more intrinsically disordered than the C-terminal segment of peripherin-2. The combination of chemical shifts with the amide-proton exchanges and signal intensities was useful for the analyses of the remaining secondary structures. The beta-structure might be more detectable by the protection of amide-proton exchange than the helical structure, although the changes in chemical shifts were sensitive for the detection of elements of both secondary structures.

Long-range effects of tag sequence on marginally stabilized structure in HIV-1 p24 capsid protein monitored using NMR.

N-terminal domain of HIV-1 p24 capsid protein is a globular fold composed of seven helices and two ß-strands with a flexible structure including the α4-5 loop and both N- and C-terminal ends. However, the protein shows a high tendency (48%) for an intrinsically disordered structure based on the PONDR VL-XT prediction from the primary sequence. To assess the possibility of marginally stabilized structure under physiological conditions, the N-terminal domain of p24 was destabilized by the addition of an artificial flexible tag to either N- or C-terminal ends, and it was analyzed using T1, T2, hetero-nuclear NOE, and amide-proton exchange experiments. When the C-terminal tag (12 residues) was attached, the regions of the α3-4 loop and helix 6 as well as the α4-5 loop attained the flexible structures. Furthermore, in the protein containing the N-terminal tag (27 residues), helix 4 in addition to the above-mentioned area including α3-4 and α4-5 loops as well as helix 6 exhibited highly disordered structures. Thus, the long-range effects of the existence of tag sequence was observed in the stepwise manner of the appearance of disordered structures (step 1: α4-5 loop, step 2: α3-4 loop and helix 6, and step 3: helix 4). Furthermore, the disordered regions in tagged proteins were consistent with the PONDR VL-XT disordered prediction. The dynamic structure located in the middle part (α3-4 loop to helix 6) of the protein shown in this study may be related to the assembly of the viral particle.

Flexible and rigid structures in HIV-1 p17 matrix protein monitored by relaxation and amide proton exchange with NMR.

The HIV-1 p17 matrix protein is a multifunctional protein that interacts with other molecules including proteins and membranes. The dynamic structure between its folded and partially unfolded states can be critical for the recognition of interacting molecules. One of the most important roles of the p17 matrix protein is its localization to the plasma membrane with the Gag polyprotein. The myristyl group attached to the N-terminus on the p17 matrix protein functions as an anchor for binding to the plasma membrane. Biochemical studies revealed that two regions are important for its function: D14-L31 and V84-V88. Here, the dynamic structures of the p17 matrix protein were studied using NMR for relaxation and amide proton exchange experiments at the physiological pH of 7.0. The results revealed that the α12-loop, which includes the 14-31 region, was relatively flexible, and that helix 4, including the 84-88 region, was the most protected helix in this protein. However, the residues in the α34-loop near helix 4 had a low order parameter and high exchange rate of amide protons, indicating high flexibility. This region is probably flexible because this loop functions as a hinge for optimizing the interactions between helices 3 and 4. The C-terminal long region of K113-Y132 adopted a disordered structure. Furthermore, the C-terminal helix 5 appeared to be slightly destabilized due to the flexible C-terminal tail based on the order parameters. Thus, the dynamic structure of the p17 matrix protein may be related to its multiple functions.

Remaining structures at the N- and C-terminal regions of alpha-synuclein accurately elucidated by amide-proton exchange NMR with fitting.

Alpha-synuclein is analyzed in physiological conditions by CLEANEX-PM methodology, in which the amide-proton exchange can be monitored at millisecond scale. The relationship between kex and [OH](-) is confirmed as a linear correlation with slope 1, indicating EX2 regime. There are significant residual structures at the N- and C-terminal regions. The structure at the C-terminal region is more stable than that of the N-terminal region. The middle part including NAC region is not completely protected. The data acquired at various pH and mixing time conditions followed by linear fitting give accurate information about residual structures.