Abstract
Human profilin-1 is a novel protein associated with a recently discovered form of familial amyotrophic lateral sclerosis. This urges the characterization of possible conformational states, different from the fully folded state, potentially able to initiate self-assembly. Under native conditions, profilin-1 is monomeric and possesses a well-defined secondary and tertiary structure. When incubated at low pH or with high urea concentrations, profilin-1 remains monomeric but populates unfolded states exhibiting larger hydrodynamic radius and disordered structure, as assessed by dynamic light scattering, far-UV circular dichroism and intrinsic fluorescence. Refolding from the urea-unfolded state was studied at equilibrium and in real-time using a stopped-flow apparatus. The results obtained with intrinsic fluorescence and circular dichroism indicate a single phase without significant changes of the corresponding signals before the major refolding transition. However, such a transition is preceded by a burst phase with an observed increase of ANS fluorescence, which indicates the conversion into a transiently populated collapsed state possessing solvent-exposed hydrophobic clusters. Kinetic analysis reveals that such state has a conformational stability comparable to that of the fully unfolded state. To our knowledge, profilin-1 is the first example of an amyloid-related protein where folding occurs in the absence of thermodynamically stable partially folded states.
Highlights
Human profilin-1, named platelet profilin, is one of the four known human profilins which acts as a complex regulator of the assembly of monomeric actin (G-actin) into filaments (F-actin)[1,2]
In order to assess whether the purified protein adopts a native structure, far-UV circular dichroism (CD) spectra of the protein in G-buffer at pH 7.3, in a solution at pH 1.7 and in another solution containing 8 M urea were acquired (Fig. 2A)
In the present manuscript we purified human profilin-1 and we first characterized the structural properties of the native, acid- and urea-unfolded states
Summary
Human profilin-1, named platelet profilin, is one of the four known human profilins which acts as a complex regulator of the assembly of monomeric actin (G-actin) into filaments (F-actin)[1,2]. The formation of intracellular inclusions by the same protein bearing the pathogenic mutations suggests that protein aggregation is a key process of the disease, highlighting the importance of elucidating its fundamental aspects to gain insight into the pathogenesis of the disorder. In this regard, fALS forms associated with mutant profilin-1 are similar to other identified forms of fALS, where aggregation occurs for the protein bearing the mutations; examples include fALS associated with mutations of superoxide dismutase 15, RNA binding protein FUS6, vesicle associated membrane protein associated protein B7, C9orf[728,9] and TAR DNA-binding protein 4310. The lack of knowledge of the folding process of profilin-1 limits our current understanding of the aggregating properties of this protein
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