Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain

Abstract

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.

Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes

“…TcTIM has different dihedral angles from TbTIM, because in both enzymes, helix 1 and helix 2 have a Pro in the middle of the sequence, which alters their structure. In contrast with other studies in which the isomerization of Pro is a factor that slows the folding process of proteins [31,32], this does not happen with TcTIM, whose reactivation is faster and more efficient and shows less aggregation than TbTIM.…”

Cardiac T-Tubule Microanatomy and Function

“…The crystal structure of the amphiphysin N-BAR domain reveals a dimeric interaction between helixes from each monomer to form a 6-helix bundle (169). With the elongated banana shape of the dimers (169) and the general function in membrane curvature formation, BIN1 has thus been referred to as the "banana" molecule (28,79,174). The interface between the monomers is largely hydrophobic, and the curvature of the dimer results from how the monomers intersect at highly conserved kinks (28,225) at the end of the 6-helix bundle.…”

“…The interface between the monomers is largely hydrophobic, and the curvature of the dimer results from how the monomers intersect at highly conserved kinks (28,225) at the end of the 6-helix bundle. These kinks are dependent on proline residues, isomerization of which represents a ratelimiting step in dimer formation (79). Domains immediately following the N-BAR in BIN1 can further strengthen its binding affinity to phospholipids for curvature formation.…”

BIN1 regulates dynamic t-tubule membrane

“…The structure of this dimer has an elongated banana shape [75]. Therefore, based on the BAR domain shape and its general function as membrane curvature protein, BIN1 has been referred to as the “banana” molecule [76–78]. The interface between the monomers is largely hydrophobic and the curvature of the dimer results from how the monomers intersect and the kinks in the helixes that heavily depend on the proline residues.…”

“…These helix kinks are highly conserved [76, 79] and reside at the end of the 6-helix bundle. A recent study shows that proline isomerization represents a rate-limiting step in proper dimer formation [78]. The N-terminal amphipatheic helix is also important for lipid binding.…”