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Transmissible spongiform encephalopathies (TSEs) are rare but fatal neurodegenerative diseases affecting human and animals. The prion protein which is the causative agent, according to “protein-only” hypothesis misfold in to rogue amyloid conformer. Despite several years of studies, the atomic structural details of the rogue conformers have not been clearly understood. This study focused on developing an in-vitro conversion method, which allows us to monitor the transition from unfolded state of prion protein to fibril state. In order to reach maximal unfolded state, we have used 8 M urea as chemical denaturant, pH 2 and prion fragment 90-230 as the model. It has been demonstrated earlier that acidic pH and mild denaturant induce the fibril formation. The mechanism underlying the structural transition from monomeric state to polymeric form is largely unknown. We have confirmed by EM and AFM that fibrils are formed in our conditions, which resemble to naturally occurring fibrils in morphologies observed. The agitation accelerates the rate of fibril formation and, which allow us to do time-resolved NMR on these preparations. The conformational flexibility is inherent to amyloid fibrils and has been observed in our preparations. We aimed to map the important segment of prion protein, which forms the rigid core in its fibrillar structured form. Our time-resolved NMR studies allowed us to monitor the changes happening from unfolded state to fibrillar state. Analysis of data identified the segment between residues 145 to 223 forming the rigid core in these fibrils, which correspond to β strand 2, helix 2 and major part of helix 3 of native prion monomeric structure. Most of the point mutations which are associated with hereditary prion disease are part of rigid core, which undergo a refolding on fibril formation. The C-terminal residues from 224 to 230 displayed peak shifting and therefore, indicate the adaptation to a fibril specific conformation. The major part of N-terminal 90-144 segment, remains dynamic, which can be understood by their accessibility to amyloid specific antibodies. This provides novel structural insight to the amyloid formation from unfolded state of prion protein fragment 90-230, which represents the proteinase-K resistant part naturally occurring prions. Earlier studies have established the core to 160-220 where hydrogen-deuterium exchange mass spectrometry or site-directed spin labeling EPR spectroscopy was used for analysis. Those studies have been initiated from either native-like or partially unfolded state of recombinant prion protein, and therefore, it is quite striking to find out that fibrils initiated from unfolded monomeric state share the same “amyloid core”. This structural insight has important implications for understanding the molecular basis of prion propagation.