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The Folding process of Human Profilin-1, a novel protein associated with familial amyotrophic lateral sclerosis.

Del Poggetto E, Chiti F, Bemporad F - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental and Clinical Biomedical Sciences, Section of Biochemistry, University of Florence, Viale Morgagni 50, I-50134, Florence, Italy.

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.

No MeSH data available.


Related in: MedlinePlus

Profilin-1 structure and purification.(A) Three-dimensional structure of native profilin-1. The structure was determined by multidimensional heteronuclear NMR spectroscopy3 and drawn with VMD for win-32 from PDB entry 1PFL. Red, blue and grey colors indicate α-helices, β-strands and loops, respectively. The positions of Trp residues are shown as sticks (Trp3, Trp31). (B) SDS-PAGE of profilin-1 after purification. An aliquot of the protein after purification (30 μg, left) and following a 3-fold dilution (10 μg, right) were loaded. Protein purity, calculated with ImageJ software, was 90–95%. (C) MALDI mass spectrometry analysis of profilin-1 after purification. The expected molecular weight for human profilin-1 devoid of the initial methionine residue is 14923 Da (Uniprot code P07737, residues 2–140).
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f1: Profilin-1 structure and purification.(A) Three-dimensional structure of native profilin-1. The structure was determined by multidimensional heteronuclear NMR spectroscopy3 and drawn with VMD for win-32 from PDB entry 1PFL. Red, blue and grey colors indicate α-helices, β-strands and loops, respectively. The positions of Trp residues are shown as sticks (Trp3, Trp31). (B) SDS-PAGE of profilin-1 after purification. An aliquot of the protein after purification (30 μg, left) and following a 3-fold dilution (10 μg, right) were loaded. Protein purity, calculated with ImageJ software, was 90–95%. (C) MALDI mass spectrometry analysis of profilin-1 after purification. The expected molecular weight for human profilin-1 devoid of the initial methionine residue is 14923 Da (Uniprot code P07737, residues 2–140).

Mentions: From a structural perspective, profilin-1 is a 139-residue protein whose three-dimensional structure has been solved with both NMR3 (PDB entry 1PFL) and X-ray crystallography (unpublished results; PDB entry 1FIK). Both structures show that it consists of an antiparallel, 7-stranded β-sheet packed between three amphipathic α-helices, two on one side of the sheet and one on the other side (Fig. 1A). This fold, which is named profilin-like according to the Structural Classification of Proteins (SCOP), is shared by roughly 25 structural families of proteins, such as the GAF-domain, the flavin-binding PAS domain, the sedlin and the YNR034W-A-like families.


The Folding process of Human Profilin-1, a novel protein associated with familial amyotrophic lateral sclerosis.

Del Poggetto E, Chiti F, Bemporad F - Sci Rep (2015)

Profilin-1 structure and purification.(A) Three-dimensional structure of native profilin-1. The structure was determined by multidimensional heteronuclear NMR spectroscopy3 and drawn with VMD for win-32 from PDB entry 1PFL. Red, blue and grey colors indicate α-helices, β-strands and loops, respectively. The positions of Trp residues are shown as sticks (Trp3, Trp31). (B) SDS-PAGE of profilin-1 after purification. An aliquot of the protein after purification (30 μg, left) and following a 3-fold dilution (10 μg, right) were loaded. Protein purity, calculated with ImageJ software, was 90–95%. (C) MALDI mass spectrometry analysis of profilin-1 after purification. The expected molecular weight for human profilin-1 devoid of the initial methionine residue is 14923 Da (Uniprot code P07737, residues 2–140).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4521207&req=5

f1: Profilin-1 structure and purification.(A) Three-dimensional structure of native profilin-1. The structure was determined by multidimensional heteronuclear NMR spectroscopy3 and drawn with VMD for win-32 from PDB entry 1PFL. Red, blue and grey colors indicate α-helices, β-strands and loops, respectively. The positions of Trp residues are shown as sticks (Trp3, Trp31). (B) SDS-PAGE of profilin-1 after purification. An aliquot of the protein after purification (30 μg, left) and following a 3-fold dilution (10 μg, right) were loaded. Protein purity, calculated with ImageJ software, was 90–95%. (C) MALDI mass spectrometry analysis of profilin-1 after purification. The expected molecular weight for human profilin-1 devoid of the initial methionine residue is 14923 Da (Uniprot code P07737, residues 2–140).
Mentions: From a structural perspective, profilin-1 is a 139-residue protein whose three-dimensional structure has been solved with both NMR3 (PDB entry 1PFL) and X-ray crystallography (unpublished results; PDB entry 1FIK). Both structures show that it consists of an antiparallel, 7-stranded β-sheet packed between three amphipathic α-helices, two on one side of the sheet and one on the other side (Fig. 1A). This fold, which is named profilin-like according to the Structural Classification of Proteins (SCOP), is shared by roughly 25 structural families of proteins, such as the GAF-domain, the flavin-binding PAS domain, the sedlin and the YNR034W-A-like families.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental and Clinical Biomedical Sciences, Section of Biochemistry, University of Florence, Viale Morgagni 50, I-50134, Florence, Italy.

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.

No MeSH data available.


Related in: MedlinePlus