02482nas a2200217   4500008004100000022001400041245010300055210006900158260000800227300001200235490000700247520173400254653002401988653002602012653003002038653001302068653001402081100002202095700002402117856012302141       2023                        eng d  a0887-358500aMultistep molecular mechanism of amyloid-like aggregation of nucleic acid-binding domain of TDP-430 aMultistep molecular mechanism of amyloidlike aggregation of nucl  cMAY  a649-6640 v913 a<p>
	TDP-43 protein is associated with many neurodegenerative diseases and has been shown to adopt various oligomeric and fibrillar states. However, a detailed kinetic understanding of the structural transformation of the native form of the protein to the fibrillar state is missing. In this study, we delineate the temporal sequence of structural events during the amyloid-like assembly of the functional nucleic acid-binding domain of TDP-43. We kinetically mapped the aggregation process using multiple probes such as tryptophan and thioflavin T (ThT) fluorescence, circular dichroism (CD), and dynamic light scattering (DLS) targeting different structural events. Our data reveal that aggregation occurs in four distinct steps-very fast, fast, slow, and very slow. The ``very fast'' change results in partially unfolded forms that undergo conformational conversion, oligomerization and bind to ThT in the ``fast step'' to form higher order intermediates (HOI). The temporal sequence of the formation of ThT binding sites and conformational conversion depends upon the protein concentration. The HOI further undergoes structural rearrangement to form protofibrils in the ``slow'' step, which, consequently, assembles in the ``very slow'' step to form an amyloid-like assembly. The spectroscopic properties of the amyloid-like assembly across the protein concentration remain similar. Additionally, we observe no lag phase across protein concentration for all the probes studied, suggesting that the aggregation process follows a linear polymerization reaction. Overall, our study demonstrates that the amyloid-like assembly forms in multiple steps, which is also supported by the temperature dependence of the kinetics.</p>
10aaggregation pathway10aamyloid-like assembly10aconformational conversion10akinetics10aoligomers1 aPillai, Meenakshi1 aJha, Santosh, Kumar  uhttp://library.ncl.res.in/content/multistep-molecular-mechanism-amyloid-aggregation-nucleic-acid-binding-domain-tdp-4302273nas a2200133   4500008004100000022001400041245016200055210006900217260000800286520167500294100002001969700002401989856012602013       2024                        eng d  a1520-610600aMulti-site red-edge excitation shift reveals the residue-specific solvation dynamics during the native to amyloid-like transition of an amyloidogenic protein0 aMultisite rededge excitation shift reveals the residuespecific s  cDEC3 a<p>
	Changes in water-protein interactions are crucial for proteins to achieve functional and nonfunctional conformations during structural transitions by modulating local stability. Amyloid-like protein aggregates in deteriorating neurons are hallmarks of neurodegenerative disorders. These aggregates form through significant structural changes, transitioning from functional native conformations to supramolecular cross-beta-sheet structures via misfolded and oligomeric intermediates in a multistep process. However, the site-specific dynamics of water molecules from the native to misfolded conformations and further to oligomeric and compact amyloid structures remain poorly understood. In this study, we used the fluorescence method known as red-edge excitation shift (REES) to investigate the solvation dynamics at specific sites in various equilibrium conformations en route to the misfolding and aggregation of the functional domain of the TDP-43 protein (TDP-43tRRM). We generated three single tryptophan-single cysteine mutants of TDP-43tRRM, with the cysteines at different positions and tryptophan at a fixed position. Each sole cysteine was fluorescently labeled and used as a site-specific fluorophore along with the single tryptophan, creating four monitorable sites for REES studies. By investigating the site-specific extent of REES, we developed a residue-specific solvation dynamics map of TDP-43tRRM during its misfolding and aggregation. Our observations revealed that solvation dynamics progressively became more rigid and heterogeneous to varying extents at different sites during the transition from native to amyloid-like conformations.</p>
1 aMore, Sonal, R.1 aJha, Santosh, Kumar  uhttp://library.ncl.res.in/content/multi-site-red-edge-excitation-shift-reveals-residue-specific-solvation-dynamics-during