We compare the folding of representative members of a protein superfamily by experiment and simulation to investigate common features in folding mechanisms. The homeodomain superfamily of three-helical, single-domain proteins exhibits a spectrum of folding processes that spans the complete transition from concurrent secondary and tertiary structure formation (nucleation-condensation mechanism) to sequential secondary and tertiary formation (framework mechanism). The unifying factor in their mechanisms is that the transition state for (un)folding is expanded and very native-like, with the proportion and degree of formation of secondary and tertiary interactions varying. There is a transition, or slide, from the framework to nucleation-condensation mechanism with decreasing stability of the secondary structure. Thus, framework and nucleation-condensation are different manifestations of an underlying common mechanism.two-state ͉ three-state ͉ framework ͉ nucleation ͉ homeodomain A Holy Grail of protein folding is to find a single mechanism. Given the diversity of protein structure and the evolutionary pressure on function and not on folding rates, a unique mechanism for folding would seem unlikely. If there are simplifying features, then small, single-domain proteins may be the most likely to exhibit them. But such proteins seem to fold by two distinct mechanisms. The 6-85 repressor fragment (1) and the engrailed homeodomain (En-HD; ref.2) seem to fold by a classical diffusion-collision mechanism (3-5) whereby secondary structural elements form independently and then dock to form the tertiary structure. Chymotrypsin inhibitor 2, on the other hand, folds by nucleation-condensation, which is characterized by concerted consolidation of secondary and tertiary interactions as the whole domain collapses around an extended nucleus (6). It has been argued on general grounds that nucleation-condensation and diffusion-collision are different manifestations of a common mechanism in which secondary structure and tertiary structure form in parallel (7,8). Nucleationcondensation reflects the situation when secondary structure is inherently unstable in the absence of tertiary interactions whereas diffusion-collision becomes more probable with increasing stability of secondary structure.Studies of the folding of point mutants of a prototype protein are essential for discovering atomic level details of folding mechanisms and kinetics. Single-point mutants may even cause gross changes in the kinetics of folding, such as the transition from three-state to two-state folding (9). But, to extrapolate a general understanding of folding mechanisms, studies on members of the same fold family (different homologues sharing the same overall topology but with different primary structures) can be useful in finding correlations between amino acid sequences and three-dimensional structures (10-16). Although there can be different folding routes through different transition states for some proteins (17), it seems that mechanisms of folding are oft...

The ability to diagnose and treat prion diseases is limited by our current understanding of the conversion process of the protein from healthy to harmful isoform. Whereas the monomeric, benign species is well characterized, the misfolded conformations responsible for infectivity and neurodegeneration remain elusive. There is mounting evidence that fibrillization intermediates, or protofibrils, but not mature fibrils or plaques, are the pathogenic species in amyloid diseases. Here, we use molecular dynamics to simulate the conversion of the prion protein. Molecular dynamics simulation produces a scrapie prion protein-like conformation enriched in ␤-structure that is in good agreement with available experimental data. The converted conformation was then used to model a protofibril by means of the docking of hydrophobic patches of the template structure to form hydrogen-bonded sheets spanning adjacent subunits. The resulting protofibril model provides a nonbranching aggregate with a 31 axis of symmetry that is in good agreement with a wide variety of experimental data; importantly, it was derived from realistic simulation of the conversion process.

Transthyretin, ␤2-microglobulin, lysozyme, and the prion protein are four of the best-characterized proteins implicated in amyloid disease. Upon partial acid denaturation, these proteins undergo conformational change into an amyloidogenic intermediate that can self-assemble into amyloid fibrils. Many experiments have shown that pH-mediated changes in structure are required for the formation of the amyloidogeneic intermediate, but it has proved impossible to characterize these conformational changes at high resolution using experimental means. To probe these conformational changes at atomic resolution, we have performed molecular dynamics simulations of these proteins at neutral and low pH. In low-pH simulations of all four proteins, we observe the formation of ␣-pleated sheet secondary structure, which was first proposed by L. Pauling and R. B. Corey [(1951) Proc. Natl. Acad. Sci. USA 37, 251-256]. In all ␤-sheet proteins, transthyretin and ␤2-microglobulin, ␣-pleated sheet structure formed over the strands that are highly protected in hydrogen-exchange experiments probing amyloidogenic conditions. In lysozyme and the prion protein, ␣-sheets formed in the specific regions of the protein implicated in the amyloidogenic conversion. We propose that the formation of ␣-pleated sheet structure may be a common conformational transition in amyloidosis.

Introduction A systematic review and meta‐analysis was performed regarding the diagnostic performance of neurofilament light chain (NfL) in CSF and blood. Methods A database search was conducted for NfL biomarker studies in the context of Alzheimer's disease (AD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) compared with controls (i.e., cognitively unimpaired, mild cognitive impairment, or disease mimics). Results In groups with a sufficient number of studies, the performance of NfL in blood and CSF was similar. Compared with disease mimics, we observed that CSF NfL had strong discriminatory power for ALS, modest discriminatory power for FTD, and no discriminatory power for AD. NfL provided the greatest separation between ALS and cognitively unimpaired controls in both the blood and CSF, followed by FTD (CSF and blood), then AD (blood and CSF). Discussion Comparable performance of CSF and blood NfL in many groups demonstrates the promise of NfL as a noninvasive biomarker of neurodegeneration; however, its utility in clinically meaningful scenarios requires greater scrutiny. Toward clinical implementation, a more comprehensive understanding of NfL concentrations in disease subtypes with overlapping phenotypes and at defined stages of disease, and the development of a harmonization program, are warranted.