Conformational properties of the aggregation precursor state of HypF-N.
The conversion of specific proteins or protein fragments into insoluble, ordered fibrillar aggregates is a fundamental process in protein chemistry, biology, medicine and biotechnology. As this structural conversion seems to be a property shared by many proteins, understanding the mechanism of this process will be of extreme importance. Here we present a structural characterisation of a conformational state populated at low pH by the N-terminal domain of Escherichia coli HypF. Combining different biophysical and biochemical techniques, including near- and far-UV circular dichroism, intrinsic and 8-anilinonaphthalene-1-sulfonate-derived fluorescence, dynamic light scattering and limited proteolysis, we will show that this state is largely unfolded but contains significant secondary structure and hydrophobic clusters. It also appears to be more compact than a random coil-like state but less organised than a molten globule state. Increase of the total ionic strength of the solution induces aggregation of such a pre-molten globule state into amyloid-like protofibrils, as revealed by thioflavin T fluorescence and atomic force microscopy. These results show that a pre-molten globule state can be, among other possible conformational states, one of the precursor states of amyloid formation. In addition, the possibility of triggering aggregation by modulating the ionic strength of the solution provides one a unique opportunity to study both the initial precursor state and the aggregation process.
Campioni
Silvia
S
Dipartimento di Scienze Biochimiche, Università di Firenze, Viale Morgagni 50, 50134 Firenze, Italy.
Mossuto
Maria F
MF
Torrassa
Silvia
S
Calloni
Giulia
G
de Laureto
Patrizia Polverino
PP
Relini
Annalisa
A
Fontana
Angelo
A
Chiti
Fabrizio
F
D000143:Acids / Q000737:chemistry; D000595:Amino Acid Sequence; D000686:Amyloidosis; D019878:Carboxyl and Carbamoyl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D012492:Salts / Q000737:chemistry; D016415:Sequence Alignment
D000143:Acids; D029968:Escherichia coli Proteins; D012492:Salts; D019878:Carboxyl and Carbamoyl Transferases; C504838:hypF protein, E coli