Protein interactions and misfolding analyzed by AFM force spectroscopy.
Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, alpha-synuclein, amyloid beta-peptide (Abeta) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for alpha-synuclein and Abeta revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly beta-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.
McAllister
Chad
C
School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA.
Karymov
Mikhail A
MA
Kawano
Yoshiko
Y
Lushnikov
Alexander Y
AY
Mikheikin
Andrew
A
Uversky
Vladimir N
VN
Lyubchenko
Yuri L
YL
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000648:ultrastructure*; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000648:ultrastructure*; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013057:Spectrum Analysis; D013696:Temperature; D051844:alpha-Synuclein / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D051844:alpha-Synuclein; C078592:amyloid beta-protein (1-40); D009113:Muramidase